Photodynamic therapy for selectively closing neovasa in eyeground tissue

ABSTRACT

A method is provided for selectively occluding neovascular vessels formed in the ocular fundus of an eye, which includes intravenously administering a mono-L-aspartyl chlorin compound to a patient; subsequently estimating an appropriate time point when the mono-L-aspartyl chlorin compound has decreased in its concentration or has been eliminated from the retinal normal vascular vessels of the patient but is still remaining at an appropriate concentration in the vascular walls of the neovascular vessels of the ocular fundus; irradiating a laser light at a 664 nm-wavelength which is initiated at the appropriate time; and using the irradiation of the laser light to target lesions comprising the neovascular vessels, at a controlled power.

TECHNICAL FIELD

This invention relates to photodynamic therapy methods for occludingselectively such neovascular vessels as formed in the ocular fundus ofeye, without involving any damage or impairment in the normal vascularvessels present in the tissues of the ocular fundus. More specifically,this invention relates to photodynamic therapy methods for occludingexclusively the choroidal neovascular vessels and/or retinal neovascularvessels, without damaging or impairing the retinal normal parenchymaltissue, the retinal normal vascular vessels and/or the choroidal normalvascular vessels of eye, which methods each comprises intravenouslyadministering mono-L-aspartyl chlorin e6 tetra-sodium salt to amammalian animal having the choroidal neovascular vessels and/or retinalneovascular vessels, and then starting to irradiate a laser light of 664nm-wavelength to the lesions comprising the neovascular vessels in theocular fundus at a certain designated fluence of the laser light and ata such time point which is chosen and designated cautiously andingeniously.

Furthermore, this invention also relates to a novel pharmaceuticalcomposition for use in a photodynamic therapy method as mentioned above,which is in the form of a single dosage unit for intravenous injection.It is well known that the choroidal neovascular vessels are formed inpatients with age-related macular degeneration and that the retinalneovascular vessels are formed in patients with proliferative diabeticretinitis.

BACKGROUND OF THE ART

It is well known that photodynamic therapy or photochemotherapy is atherapeutic method which comprises administering a photosensitizer to apatient having pathological lesions, followed by irradiating a light ofa wavelength active to excite the photosensitizer, to the pathologicallesions where the administered photosensitizer has accumulated.

In a method of photodynamic therapy (abbreviated as PDT hereinafter)which is applied to the therapeutic treatment of cancer or tumor, thereis administered such a photosensitizer which is capable of accumulatingin the cancer or tumor tissue, particularly in the endothelial celllayers of the neovascular vessels present in the cancer or tumor tissue.Subsequently, the cancer tissue or the vascular walls containing thephotosensitizer accumulated therein is irradiated with an exciting laserlight active to excite photochemically the photosensitizer, so that thethus excited photosensitizer can further excite the oxygen moleculespresent in the surrounding regions around the photosensitizer. Throughthe excitation of the oxygen molecules, singlet oxygen (active oxygen)can be generated. Due to the cytotoxicity of the singlet oxygen, thecancer or tumor cells and the endothelial cells of the neovascularvessels containing the excited photosensitizer can be necrosed.

At an early development stage of PDT, hematoporphyrin derivatives suchas photofrin, rose bengal and others were used as first-generationphotosensitizers. The first-generation photosensitizers for use in PDThad such drawbacks that they have a poor selectivity to accumulate inthe target tumor cells while they can readily accumulate in the normalcells, and that the first-generation photosensitizers can additionallyinvolve a long-term phototoxicity in the skin of the patients [see, forexample, Cancer Res., Vol. 38, pp. 2628–2635 (1978); and Cancer Res.,Vol. 52, pp. 924–930 (1992)].

In recent years, the second-generation photosensitizers have beendeveloped for PDT of cancer. Benzoporphyrin derivatives, mono-L-aspartylchlorin e6 and others have been known as the second-generationphotosensitizers [see, for example, a report of Kessel et al., entitled“Photodynamic therapy and bio-medical lasers”, pp. 526–530 (1992),Elsevier Science Publishers, Co., Amsterdam; and a report of Nelson etal., Cancer Res., Vol. 47, pp. 4681–4685 (1987)]. Mono-L-aspartylchlorin e6 is one of the known fluorescent tetrapyrrol derivatives. Theproperties of mono-L-aspartyl chlorin e6 which is present in the cellsin vivo have been examined in detail by W. G. Roberts [see a report ofW. G. Roberts, entitled “Role of Neovasculature and VascularPermeability on the Tumor Retention of Photodynamic Agents”, CancerRes., Vol. 52, pp. 924–930 (1992)]. Based on the experiments of W. G.Roberts et al., with using chicken chorioallantoic membrane (abbreviatedas CAM hereinafter) which is a tissue formed of the differentiation-typeneovascular vessels of fertilized chicken egg, it was reported that theproperty of a photosensitizer to be selectively uptaken in the cancercells and be accumulated therein can vary depending on the sort of thephotosensitizer. In the above-mentioned reports, it is stated thatmono-L-aspartyl chlorin e6 possesses a significantly higher selectivityto accumulate in the cancer cells, as compared with chlorin e6 anduro-porphyrin. Mono-L-aspartyl chlorin e6 or a salt thereof is able toabsorb well a light of 664 nm-wavelength which is permeable deeply intoanimal tissue, so that the mono-L-aspartyl chlorin e6 compound can bephoto-excited well in vivo after administration thereof. Additionally,it has been confirmed from some experiments with mice thatmono-L-aspartyl chlorin e6 or a salt thereof can be metabolized and becleared or eliminated from a living body of the host animal at aclearance speed of 10 times higher or more than that of the previouslyknown hematoporphyrin derivatives, and that the concentration ofmono-L-aspartyl chlorin e6 present in the plasma can decrease to aconcentration of 1/100-fold of the initial concentration thereof in 10hours after the first time of the intravenous administration ofmono-L-aspartyl chlorin e6 [see the report of Kessel, et al., supra.,entitled “Photodynamic therapy and Biomedical lasers”, pp. 526–530(1992)]; and a report of C. J. Gomer and A. Ferrario et al., CancerRes., Vol. 50, pp. 3985–3990 (1990)].

The above-mentioned experiments made by W. G. Roberts et al., with usingchicken chorioallantoic membrane (CAM) have revealed thatmono-L-aspartyl chlorin e6 tetra-sodium salt is able to be moreselectively uptaken and accumulated at a higher concentration in theactively growing cells of the neovascular vessels in CAM, than suchconcentration at which the mono-L-aspartyl cholorin e6 salt can beuptaken and accumulated in the cells of the normal vascular vessels.

It has been indicated that mono-L-aspartyl chlorin e6 or a salt thereofhas further characteristic properties that it is able to bind readily tothe blood albumin and is hardly diffused in the normal tissue or normalvascular vessels having barriers, because mono-L-aspartyl chlorin e6 ora salt thereof has a low lipophilicity, and that the intracellularmigration of the mono-L-aspartyl chlorin e6 compound will occur notthrough the physical diffusion but through the cellular endocytosis orcellular absorption.

Incidentally, Japanese Patent Publications Nos. 88902/1994 and89000/1994 as well as their corresponding U.S. Pat. Nos. 4,675,338 and4,693,885 describe, for example, mono-L-aspartyl chlorin e6 andmono-L-glutamyl chlorin e6 or salts thereof. And, these patentsmentioned above describe also that the tetrapyrrol derivatives may beused as a photosensitizer for diagnosis and therapeutic treatment oftumor. In the aforementioned Japanese patents and USA patents, it isdescribed that a fluorescent tetrapyrrol derivative which has beenaccumulated in the tumor tissue after the administration thereof can bephoto-excited by irradiation of a potent light such as laser beam, sothat the tetrapyrrol derivative so photo-excited can exert a necroticaction on the tumor cells.

There are known some experiments where the choroidal neovascular vesselsof a monkey eye were destroyed by PDT with using rose bengal as aphotosensitizer [Arch. Ophthalmol., Vol. 111, June/1993, pp. 855–860].There are known additional experiments where the choroidal neovascularvessels of a monkey eye were occluded by PDT with using benzoporphyrinderivative mono-acid (Verteporfin as the photosensitizer) [Arch.Ophthalmol., Vol. 113, June/1995, pp. 810–818].

Furthermore, U.S. Pat. Nos. 5,705,518 and 5,770,619 of Richer et al.,describe a PDT experiment where a photosensitizer, benzoporphyrinderivative mono-acid ring A (BPD-MA) is prepared as its liposome and isintravenously administered to a mouse having transplanted M-1 tumor,followed by irradiating an exciting laser beam to the mouse. Based onthese experiments of Richter et al, there is proposed a method fordestroying or impairing an area of neovascularization, which comprisestranscutaneously irradiating said area with a laser light before anadministered photosensitizer has permeated into dermal tissue or othernormal tissues, so that the dermal phototoxicity can be avoided. Inthese patents of U.S. Pat. Nos. 5,705,518 and 5,770,619, Richter et al.refer to mono-L-aspartyl chlorin e6 as one example of thephotosensitizer, and they additionally refer to that the method asproposed by Richer et al is possible to be applied to the destruction orimpairment of the area of neovascularization as formed in the eye.However, there is nowhere disclosed any experimental Example which toshow that any practical application of the method of Richer et al. wasmade to the field of ophthalmology.

The present inventors, namely Dr. Mori and Dr. Yoneya et al., previouslyproposed a method for photochemotherapeutically occluding neovascularvessels in the ocular fundus, and this proposed method was based on theresults of their experiments wherein the normal vascular vessels in theocular fundus of a normal pigmented rabbit were occluded, when the PDTwith mono-L-aspartyl chlorin e6 tetra-sodium salt as a photosensitizerwas applied to the normal vascular vessels (see U.S. Pat. No.5,633,275). Furthermore, Dr. Mori and Dr. Yoneya et al., have carriedout some experiments in which there are diagnosed such impairment of theretina and such occlusion of choroidal normal vascular vessel which hadbeen involved when the PDT with mono-L-aspartyl chlorin e6 tetra-sodiumsalt as a photosensitizer was applied to the choroidal normal vascularvessels in the eye of Japanese monkey, with irradiating a laser light of664 nm-wavelength at a fluence of 7.5 J/cm² or less [Ophthalmology, Vol.106, No. 7, pp. 1384–1391 (July, 1999)].

On the other hand, it is known that the neovascularization occurring inthe ocular fundus tissue can severely damage the visual functions. Forexample, the choroidal neovascularization as involved by the age-relatedmacular degeneration is a major cause for the intermediate blindness.Due to the occurrence of the choroidal neovascularization in theage-related macular degeneration, there are induced subretinal bleedingand subretinal exudation as well as retina detachment and fibrousproliferation, which can bring about a visual deterioration andoccasionally blindness. Furthermore, it is known that when diabetesmellitus has progressed, a proliferative neovascularization is incurredin the retina, resulting in an onset of the proliferative diabeticretinitis and leading sometimes to blindness.

Hithertobefore, clinical therapeutic treatment of theneovascularization, which has occurred in the ocular fundus due to theage-related macular degeneration or the proliferative diabeticretinitis, has been done usually by making photo-coagulation of theneovascular vessels with irradiation of a laser light having a thermalaction. It has been known that the photo-coagulation method made in theabove prior art has a drawback that the thermal action of the laserlight as employed can destroy even the surrounding normal tissues,whereas the neovascular vessels themselves can be occluded well by thethermal coagulation of them. However, if a selective occlusion of theneovascular vessels as formed in the ocular fundus could be madefeasible by means of PDT with using a photosensitizer and a laser lightirradiation, it can be expected that a satisfactory therapeutic methodfor selectively occluding the neovascular vessels in the ocular funduswould be developed and provided. In the past, therefore, a great numberof research works have been carried out for further ophthalmologicalapplication of PDT.

DISCLOSURE OF THE INVENTION

An object of this invention is to provide such novel, photodynamictherapy methods which are able to selectively occlude the choroidalneovascular vessels and/or the retinal neovascular vessels withoutinvolving any damage or impairment in the retinal parenchymal tissue aswell as in the retinal normal vascular vessels and the choroidal normalvascular vessels, while there is adopted here such PDT method comprisingadministrating mono-L-aspartyl chlorin e6 tetra-sodium salt as thephotosensitizer and irradiating the laser light of 664 nm-wavelength,and which novel photodynamic therapy method can also be designed to beoperated in the clinical practice with safety.

The present inventors have now carried out a series of experimentswherein mono-L-aspartyl chlorin e6 tetra-sodium salt (sometimesabbreviated as NPe6 hereinafter) is intravenously injected at varyingdoses to test animals of monkey having neovasculature lesions formed ofsuch experimental choroidal neovascular vessels (abbreviated as CNVsometimes hereinafter) which have been produced according to amodification of the Ryan's method of irradiating an argon laser lightonto the ocular fundi of monkeys [Arch. Ophthalmol., Vol. 100, pp.1804–1809 (1982)], and wherein observations are made of time-dependentchanges in the concentration of the administered NPe6 as uptaken anddistributed in the normal retinal parenchymal tissue, also in thevascular walls of the retinal normal vascular vessels and in thevascular walls of the CNV present in said neovasculature lesions formedof the experimental CNV, after the intravenous administration of NPe6was done, and wherein there are utilized, as a guiding sign, thetime-dependent changes in the intensity of a red fluorescence which canbe emitted from NPe6 as excited under irradiation of a laser light of488 nm-wavelength.

From the results of these experiments made by the present inventors, ithas now been found that the administered NPe6 can never permeate intothe normal parenchymal tissue of the ocular retina. Additionally, it hasalso now been found that a significant quantity of the administered NPe6can be uptaken and accumulated in the vascular walls of the retinalnormal vascular vessels, before the administered NPe6 is uptaken in thevascular walls of CNV, and that a significant quantity of NPe6 can thenpermeate into the vascular walls of CNV, while the administered andaccumulated NPe6 can subsequently continue to be cleared or eliminatedfrom the retinal normal vascular walls at a higher clearance speed thanfrom the vascular walls of CNV.

It is also now revealed that, during the course where NPe6 has once beenuptaken and accumulated in the vascular walls of the retinal normalvascular vessels and is subsequently cleared or eliminated out of saidvascular walls of the normal retinal vascular vessels, the NPe6 cancommence to be uptaken into and accumulated in the vascular walls of thechoroidal neovascular vessels (CNV), and that the concentration of NPe6as uptaken and accumulated and remaining in the vascular walls of CNVcan reach a peak of its concentration in about 20 minutes or thereaboutafter the time of the intravenous injection of NPe6. Furthermore, it isalso now found that, in the course of the clearance or elimination ofNPe6 from the vascular walls of the retinal normal vascular vessels, andalso during a time period as lapsed after the observed completeclearance or elimination of NPe6 in the vascular walls of the retinalnormal vascular vessels, NPe6 can continue to permeate and remain at asignificant concentration of NPe6 preferentially in the vascular wallsof CNV.

It is now further found that, after the administration of NPe6 to themonkeys under test, the time-dependent change in the concentration ofNPe6 as uptaken and distributed in the vascular walls of the retinalnormal vascular vessels can vary dependently on the doses of NPe6administered, and that the time-dependent change in the concentration ofNPe6 as uptaken and remaining in the vascular walls of CNV can also varydependently on the doses of NPe6 administered. When the dose of theintravenously injected NPe6 is controlled within a range of 0.5 mg/kg to10 mg/kg, it can also be found now that the concentration of the NPe6remaining in the vascular walls of CNV can be retained at a remarkablyhigher value than that of the concentration of NPe6 remaining in thevascular walls of the retinal normal vascular vessels, as long as thedetermination of the NPe6 concentration is done at a certain chosen timepoint or time points after the intravenous injection of NPe6, thoughsaid time point(s) of determining the NPe6 concentration is or arealtered with the doses of NPe6 administered; and further it is now foundthat the concentration of NPe6 remaining in the vascular walls of CNVcan be retained at a significantly high value even after NPe6 had beencompletely or almost completely cleared or eliminated from the vascularwalls of the retinal normal vascular vessels.

The present inventors have now found additionally that, when a laserlight of 664 nm-wavelength is started to be irradiated to the CNVpresent in said neovasculature lesions, either at such a time point atwhich the concentration of NPe6 remaining in the vascular walls of theretinal normal vascular vessels has reached a value of the NPe6concentration significantly lower than the value of the concentration ofNPe6 remaining in the vascular walls of CNV, or at such a time point atwhich a significant concentration of NPe6 can be observed to remainstill in the vascular walls of CNV even after NPe6 can be observed tohave been cleared or eliminated completely from the retinal normalvascular vessels; and also when the above-mentioned irradiation of thelaser light of 664 nm-wavelength to the CNV in the neovasculaturelesions is effected at an ingeniously adjusted fluence of said laserlight, it is feasible that the vascular endothelial cells of the CNV sotreated can successfully be destroyed photochemically and the CNV canthus be occluded preferentially or selectively, without involving anyadverse effect in the retinal parenchymal tissue, the retinal normalvascular vessels and the choroidal normal vascular vessels.

In contrast, it has now been found that, if the laser light of 664nm-wavelength is started to be irradiated at an inappropriate time pointafter the intravenous injection of NPe6, and/or if the fluence of saidlaser light is adjusted to an insufficient value or to an excessivelyhigh value of the fluence of the laser light even with the irradiationof said laser light being started at an appropriate time point, it isimpossible to achieve the selective occlusion of CNV as desired.

Thus, it has now been found that, for instance, if the irradiation ofthe laser light of 664 nm-wavelength is started toward theneovasculature lesions at such an inappropriate time point when NPe6 isstill remaining at a significantly high concentration of NPe6 in thevascular walls of the retinal normal vascular vessels, there can beinvolved undesired damage and undesired occlusion also in the retinalnormal vascular vessels and the choroidal normal vascular vessels whichare adjacent to said neovasculature lesions as exposed to said laserlight. Further, it is useless that the irradiation of the laser light of664 nm-wavelength is started at such a time point when the concentrationof NPe6 remaining in the vascular walls of the neovasculature lesionshas decreased to an inappropriately lowered value.

The present inventors have now found that, in order to achieve theobject of this invention for attaining the selective occlusion of CNV,it is necessary that the laser light of 664 nm-wavelength is started tobe irradiated at such a chosen appropriate time point when theconcentration of NPe6 distributed and remaining in the vascular walls ofthe retinal normal vascular vessels has either reached a value of theNPe6 concentration sufficiently lower than the value of theconcentration of NPe6 remaining in the vascular walls of CNV, or hasreached a value of zero or a value close to zero; and also it isnecessary that the fluence of the laser light of 664 nm-wavelength to beirradiated is controlled to an appropriate value. It may be consideredthat the above-mentioned various findings as obtained by the presentinventors from the above-mentioned PDT experiments with monkeys maycommonly be applicable to the PDT technique which is to be carried outto achieve a selective occlusion of the ocular neovascular vessels ofhuman beings, because it is known that the structure of the ocularfundus tissue in the monkey eye is very similar to the structure of theocular fundus tissue in the human eye. This invention has beenaccomplished on the basis of the above various findings of the presentinventors.

In a first aspect of the invention, therefore, there is provided aphotodynamic therapy method for occluding selectively such choroidalneovascular vessels and/or retinal neovascular vessels as formed in amammalian animal having an ocular fundus tissue comprising the retinalnormal parenchymal tissue, the retinal normal vascular vessels and thechoroidal normal vascular vessels lying under the retina, as well aschoroidal neovascular vessels and/or retinal neovascular vessels, bydeveloping actions of the photochemical reaction of an administeredphotosensitizer as excited with an irradiating laser light, but withoutinvolving a substantial damage or impairment in the retinal normalparenchymal tissue, the retinal normal vascular vessels and thechoroidal normal vascular vessels, characterized in that the methodcomprises:

-   (a) administering intravenously mono-L-aspartyl chlorin e6 or a    pharmaceutically acceptable salt thereof, particularly    mono-L-aspartyl chlorin e6 tetra-sodium salt as the photosensitizer    to the animal at a dose of 0.5 mg/kg to 10 mg/kg (as calculated on    the weight basis of mono-L-aspartyl chlorin e6 tetra-sodium salt) at    a vein of the animal;-   (b) allowing that the mono-L-aspartyl chlorin e6 or a salt thereof,    particularly mono-L-aspartyl chlorin e6 tetra-sodium salt    administered as the photosensitizer, which is carried along by the    blood streams circulating in the ocular retinal central artery and    ciliary artery, is uptaken in the endothelial cell layers of the    retinal normal vascular vessels and also in the endothelial cell    layers of the choroidal normal vascular vessels as well as in the    endothelial cell layers of the retinal neovascular vessels and/or    the endothelial cell layers of the choroidal neovascular vessels,    and allowing that the administered photosensitizer is then    distributed and accumulated in and being eliminated or cleared from    the vascular walls of said normal vascular vessels and said    neovascular vessels;-   (c) subsequently irradiating and scanning a laser light of 488    nm-wavelength at a sufficient fluence of said laser light,    intermittently or continuously, through the surface of ocular    cornea, pupil, lens and vitreous fluid of the eye, to and on said    ocular fundus tissue of the animal, so as to induce emission of a    red fluorescence from the photo-excited mono-L-aspartyl chlorin e6    having distributed in the vascular walls of said normal vascular    vessels and of said neovascular vessels in said ocular fundus    tissue; and further observing intermittently or continuously the    intensity of the red fluorescence which is emitted from the    photosensitizer having been exposed to the laser light of 488    nm-wavelength and having distributed in the vascular walls of the    retinal normal vascular vessels as well as in the vascular walls of    the choroidal neovascular vessels and/or the vascular walls of the    retinal neovascular vessels, with using a fluorescence ocular fundus    angiography apparatus or a fluorescence microscope for funduscopy,    for this observation;-   (d) using said fluorescence ocular fundus angiography apparatus or    fluorescence microscope for funduscopy during the irradiation of the    488 nm-wavelength laser light, to estimate such a time point at    which, in the course of the elimination or clearance of the    photosensitizer out of the vascular walls of the retinal normal    vascular vessels and the choroidal normal vascular vessels, the    intensity of the red fluorescence emitted from the photosensitizer    present in the vascular walls of the retinal normal vascular vessels    under observation can be observed to have decreased to a lowered    value of about one-half-folds or less, particularly ⅓-folds or less    of the intensity of the red fluorescence emitted from the    photosensitizer remaining in the vascular walls of the neovascular    vessels under observation; and thus to estimate such time point at    which it can be revealed from the observation of the decrease in the    intensity of the emitted red fluorescence by means of said    fluorescence ocular fundus angiography apparatus or fluorescence    microscope for funduscopy that the concentration of the    photosensitizer present in the vascular walls of the retinal normal    vascular vessels under observation has decreased to a lowered value    of about one-half-folds or less, particularly ⅓-folds or less of the    concentration of the photosensitizer remaining in the vascular walls    of the neovascular vessels under observation;-   (e) at the time point so estimated in the above step (d) or within a    time of 1 to 10 minutes from the so estimated time point; starting    to irradiate a laser light of 664 nm-wavelength in the form of a    thin laser beam, via the cornea, pupil and vitreous fluid of the    eye, exclusively to such targeted lesions comprising the    neovasculature formed of said neovascular vessels in the ocular    fundus, in such a way that said laser light of 664 nm-wavelength is    irradiated at a fluence of said laser light necessary to excite the    photosensitizer remaining in the vascular walls of the neovascular    vessels; and-   (f) subsequently permitting that the lumens of the neovascular    vessels contained in said lesions as irradiated with the laser light    of 664 nm-wavelength are occluded by the developed actions of the    photochemical reaction of the laser-excited photosensitizer    remaining in the vascular walls of said neovascular vessels, whereby    a selective occlusion of the choroidal neovascular vessels and/or    the retinal neovascular vessels is achieved.

According to the first aspect method of this invention, thephotosensitizer may usually be generally administered intravenously at avein other than the ocular veins but may be administered also at anintraocular vein if a possible means is available therefor.

Mono-L-aspartyl chlorin e6 is a compound which may be produced by themethod described in Example 19 of the specification of U.S. Pat. No.4,675,338 described above. Mono-L-aspartyl chlorin e6 is a substancehaving the chemical structure represented by the formula (A):

In this invention, the term “mammalian animal” means an animal,including humans. Generally, the mono-L-aspartyl chlorin e6 as used inthis invention may be in the form of a salt thereof with a base. Thesalt with a base may include, for example, the salts with sodium,potassium, calcium, magnesium, ammonium, triethylammonium,trimethylammonium, morpholine and piperidine.

When acute toxicity of mono-L-aspartyl chlorin e6 was tested, it is alsofound that the LD₅₀ of mono-L-aspartyl chlorin e6 tetra-sodium salt inCD-1 (male) mice is 164 mg/kg. From phototoxicity test, it is also foundthat mono-L-aspartyl chlorin e6 tetra-sodium salt has got an approval asa safe compound which dose not involve any side-reaction of inducingerythema, edema and others.

For a laser source of irradiation of the laser light of 664nm-wavelength which is to be used in the method of this invention, it ispossible to use a semiconductors for continuous potent laser oscillationwhich is provided with optical filters; or excited dyes; or other laserdelivery systems. The laser irradiation source may desirably be such onewhich is capable of oscillating a laser beam of a wavelength of 620 to760 nm at an irradiance of 10 to 1500 mW/cm². Several of laseroscillators which are currently commercially available may satisfy theaforementioned criteria for the laser oscillation.

According to the method of the first aspect of this invention, thefluorescence, which is emitted from mono-L-aspartyl chlorin e6 asdistributed and remaining in the vascular walls after the administrationthereof, may be observed by using, for example, a scanning laserophthalmoscope (abbreviated as SLO; manufactured by Rodenstock, Co.),with exciting the mono-L-aspartyl chlorin e6 under irradiation of anargon laser light of 488 nm-wavelength.

In the method of the first aspect of this invention, it is feasiblethat, in the step (e) of the method, the irradiation of the laser lightof 664 nm-wavelength via the surface of cornea, pupil and vitreous fluidof the eye exclusively to the targeted lesions comprising theneovasculature formed of the neovascular vessels in the ocular fundus isstarted at such a time point when a time period of 10 to 70 minutes haslapsed after the time of the intravenous injection of mono-L-aspartylchlorin e6 or a salt thereof, particularly mono-L-aspartyl chlorin e6tetra-sodium salt; provided that said time point is also the time pointwhen it can be estimated from the observation of the decreases in theintensity of the red fluorescence as emitted from the photosensitizerremaining in the vascular walls by excitation of said photosensitizerwith the irradiation of the laser light of 488 nm-wavelength, with usingthe fluorescence ocular fundus angiography apparatus or the fluorescencemicroscope for funduscopy for the purpose of said observation, that theconcentration of said photosensitizer present in the vascular walls ofthe retinal normal vascular vessels has decreased just to a loweredvalue of about one-half-folds or less of the concentration of thephotosensitizer remaining in the vascular walls of the choroidalneovascular vessels and/or the vascular walls of the retinal neovascularvessels.

In the method of the first aspect of this invention, it is also feasiblethat, in the step (d) of the method, there is estimated such a timepoint when a time period of 20 to 70 minutes has lapsed after the timeof the intravenous injection of mono-L-aspartyl chlorin e6 or a saltthereof, particularly mono-L-aspartyl chlorin e6 tetra-sodium salt;provided that said time point is also such time point when it can berevealed from the observation with the fluorescence ocular fundusangiography apparatus or fluorescence microscope for funduscopy duringthe irradiation of the 488 nm-wavelength laser light, that the intensityof the red fluorescence emitted from the said photosensitizer remainingin the vascular walls of the choroidal neovascular vessels and/or theretinal neovascular vessels has reached its peak value under theirradiation of the laser light of 488 nm-wavelength; and provided thatsaid time point is further such time point when it is additionallyobserved by said fluorescence ocular fundus angiography apparatus orfluorescence microscope for funduscopy that the red fluorescence asemitted from the photosensitizer still remaining in the vascular wallsof the retinal normal vascular vessels has disappeared completely duringthe irradiation of the laser light of 488 nm-wavelength made at asufficiently high fluence of said laser light. And, it is then feasiblethat just at the time point so estimated in the above, the laser lightof 664 nm-wavelength is started in the step (e) of the method to beirradiated through the surface of cornea, pupil and vitreous fluid ofthe eye exclusively to the targeted lesions comprising theneovasculature formed of the neovascular vessels in the ocular fundus.

In the method of the first aspect of this invention, it is preferredthat the laser light of 664 nm-wavelength, which is irradiated to thetargeted neovasculature lesions comprising the neovascular vessels inthe ocular fundus, has an irradiance of 10 to 1500 mW/cm², preferably0.5 to 0.8 W/cm² on the top retina face, as measured at the cornealsurface by means of an optical power meter, and said laser light of 664nm-wavelength is irradiated for a duration of 10 to 300 seconds and at afluence of the laser light in a range of 7.0 J/cm² to 250 J/cm²,preferably 7.5 J/cm² to 205 J/cm²; provided that said fluence of thelaser light is evaluated by multiplying the laser irradiance (in W/cm²)by the duration of the laser irradiation (in seconds), whereupon thelower is the dose of mono-L-aspartyl chlorin e6, the fluence of thelaser light to be irradiated may be controlled to be the higher, so asto achieve the selective occlusion of the neovascular vessels present inthe targeted neovasculature lesions.

In the method of the first aspect of this invention, it is also possiblethat the laser light of 664 nm-wavelength is started in the step (e) ofthe method to be irradiated at such a time point when a time period of20 to 70 minutes has lapsed after the time of the intravenousadministration of mono-L-aspartyl chlorin e6 or a salt thereof,particularly mono-L-aspartyl chlorin e6 tetra-sodium salt, as long asthe chlorin e6 compound is given at a dose of 0.5 mg/kg to 10.0 mg/kg[as calculated on the weight basis of mono-L-aspartyl chlorin e6tetra-sodium salt, namely NPe6; though the same way of this calculationis applied hereinafter), and that the fluence of the laser light of 664nm-wavelength to be irradiated is controlled within a range of 7 J/cm²to 205 J/cm². It is further possible that the laser light of 664nm-wavelength is started in the step (e) of the method to be irradiatedat such a time point when a time period of 20 to 30 minutes has lapsedafter the time of the intravenous administration of mono-L-aspartylchlorin e6 or a salt thereof, particularly mono-L-aspartyl chlorin e6tetra-sodium salt, as long as the chlorin e6 compound is given at a doseof 0.5 mg/kg to 0.9 mg/kg (as calculated on the weight basis of NPe6),and that the fluence of the laser light of 664 nm-wavelength to beirradiated is controlled within a range of 175 J/cm² to 205 J/cm².

In the method of the first aspect of this invention, it is furthermorepossible that the laser light of 664 nm-wavelength is started in thestep (e) of the method to be irradiated at such a time point when a timeperiod of 30 to 60 minutes has lapsed after the time of the intravenousadministration of mono-L-aspartyl chlorin e6 or a salt thereof,particularly mono-L-aspartyl chlorin e6 tetra-sodium salt, as long asthe chlorin e6 compound is given at a dose of 1 mg/kg to 1.9 mg/kg (ascalculated on the weight basis of NPe6), and that the fluence of thelaser light of 664 nm-wavelength to be irradiated is controlled within arange of 30 J/cm² to 175 J/cm², preferably 34 J/cm² to 171 J/cm².

In the first aspect method of this invention, it is further possiblethat the laser light of 664 nm-wavelength is started in the step (e) ofthe method to be irradiated at such a time point when a time period of60 minutes has lapsed after the time of the intravenous administrationof mono-L-aspartyl chlorin e6 or a salt thereof, particularlymono-L-aspartyl chlorin e6 tetra-sodium salt, as long as the chlorin e6compound is given at a dose of 2 mg/kg to 9.5 mg/kg (as calculated onthe weight basis of NPe6), and that the fluence of the laser light of664 nm-wavelength to be irradiated is controlled within a range of 30J/cm² to 45 J/cm².

In the first aspect method of this invention, besides, it is possiblethat the laser light of 664 nm-wavelength is started in the step (e) ofthe method to be irradiated at such a time point when a time period of60 to 70 minutes has lapsed after the time of the intravenousadministration of mono-L-aspartyl chlorin e6 or a salt thereof,particularly mono-L-aspartyl chlorin e6 tetra-sodium salt, as long asthe chlorin e6 compound is given at a dose of 9.5 mg/kg to 10 mg/kg (ascalculated on the weight basis of NPe6), and that the fluence of thelaser light of 664 nm-wavelength to be irradiated is controlled within arange of 7 J/cm² to 9 J/cm², preferably 7.5 J/cm² to 8 J/cm².

In the method according to the first aspect of this invention, themammalian animal to be treated may be a human having suffered fromage-related macular degeneration with the choroidal neovascular vessels,or a human having suffered from proliferative diabetic retinitis withproliferative neovascular vessels in the retina.

In the first aspect method of this invention as described hereinbefore,the intensity of the red fluorescence emitted from mono-L-aspartylchlorin e6, particularly NPe6 having distributed and remaining in thevascular walls of the retinal normal vascular vessels is observed, incomparison with the concurrently observed intensity of the redfluorescence emitted from mono-L-aspartyl chlorin e6 having distributedand remaining in the vascular walls of CNV in the neovasculaturelesions, in such a manner that these observations are made underirradiation of an infrared light of 488 nm-wavelength and with using afluorescence ocular fundus angiography apparatus or a similar means.Thereby, it can be detected that a difference exists normally betweenthe concentration of mono-L-aspartyl chlorin e6 having distributed andremaining in the vascular walls of the retinal normal vascular vesselsand the concentration of mono-L-aspartyl chlorin e6 having distributedand remaining in the vascular walls of CNV, whereby it can be estimatedwhen is an appropriate time point at which the irradiation of the laserlight beam of 664 nm-wavelength is to be started. However, in fact, thered fluorescence emitted from the mono-L-aspartyl chlorin e6 havingdistributed and remaining in the vascular walls of the normal vascularvessels, as well as the red fluorescence emitted from themono-L-aspartyl chlorin e6 having distributed and remaining in thevascular walls of CNV are both emitted at a very low intensity evenunder the irradiation of the infrared light of 488 nm. Therefore, aconsiderable skillness is needed usually for the observing persons inorder to estimate the time-dependent changes in the intensity of the redfluorescence of mono-L-aspartyl chlorin e6 present in the vascularwalls. In particular, when mono-L-aspartyl chlorin e6 is given at a lowdose of 0.5 mg/kg or 1 mg/kg, it is needed to use various measures suchthat the sensitivity of the fluorescence ocular fundus angiographyapparatus is elevated to a maximum.

For these reasons, the present inventors have now further made anotherresearches in order to find out and examine if it is possible to developa further simpler process which can determine the concentrations ofmono-L-aspartyl chlorin e6 having distributed and remaining in thevascular walls of the retinal normal vascular vessels and of theneovascular vessels, in place of the above-mentioned process comprisingobserving the red fluorescence which is emitted from the mono-L-aspartylchlorin e6 having distributed and remaining in the vascular walls of theretinal normal vascular vessels and of the neovascular vessels in theocular fundus. Upon making these another researches, the presentinventors have paid their attention on an infrared fluorescentsubstance, indocyanine green (abbreviated as ICG) which isconventionally and frequently used in the prior art diagnosis method ofmaking the infrared fluorescence fundus angiography of the neovascularvessels in the ocular fundus of a patient with age-related maculardegeneration. ICG has the same biochemical properties as those of NPe6in the following points, that is, (i) ICG is hydrophilic similarly toNPe6; (ii) ICG has a molecular weight almost equal to NPe6; and (iii)ICG has a high affinity to lipoprotein. These indicated biochemicalproperties (i) to (iii) for a medicinal compound are known to playsignificant roles in the pharmacodynamics of the medicinal compound whenadministered to patients. Additionally, it is known that ICG can emit asufficiently stronger fluorescence than NPe6.

The present inventors have now carried out a series of furtherexperiments with using test monkeys which have the experimental CNV inthe retina, and which were used in the aforesaid experiments of makingthe intravenous administration of NPe6. In this series of furtherexperiments, indocyanine green (ICG) is intravenously injected to thetest monkeys at a dose of 0.5 mg/kg to 1 mg/kg, which dose isconventional in the routine method of making the fluorescence fundusangiography of the ocular fundus of human patients. Then, the presentinventors have examined the time-dependent change in the intensity ofinfrared fluorescence of ICG as distributed and remaining in thevascular walls of the retinal normal vascular vessels in the monkeys, incomparison with the time-dependent change in the intensity of infraredfluorescence of ICG as distributed and remaining in the vascular wallsof CNV in the neovasculature lesions in the ocular fundus of the testmonkeys, according to the routine diagnosis method of making theinfrared fluorescence fundus angiography of the ocular fundus of humanpatients.

The above-mentioned series of the further experiments has now been foundto reveal that the manner or pattern of the time-dependent change in theintensity of the infrared fluorescence of ICG as distributed andremaining in the vascular walls of the retinal normal vascular vessels,as well as the manner or pattern of the time-dependent change in theintensity of the infrared fluorescence of ICG as distributed andremaining in the vascular walls of CNV in the neovasculature lesions inthe ocular fundus of the test monkeys having received the intravenousinjection of ICG at its dose of 0.5 mg/kg to 1 mg/kg, are, respectively,very much well analogous to the manner or pattern of the time-dependentchange in the intensity of the red fluorescence of NPe6 as distributedand remaining in the vascular walls of the retinal normal vascularvessels, as well as the manner or pattern of the time-dependent changein the intensity of the red fluorescence of NPe6 as distributed andremaining in the vascular walls of CNV in the neovasculature lesions inthe ocular fundus of the test monkeys having received the intravenousinjection of NPe6 at its dose of 05 mg/kg to 1 mg/kg.

Accordingly, the present inventors have thus presumed that the patternsor manners of the time-dependent changes in the intensity of theinfrared fluorescence of ICG as distributed and remaining in thevascular walls of the retinal normal vascular vessels and the patternsor manners of the time-dependent change in the intensity of the infraredfluorescence of ICG as distributed and remaining in the vascular wallsof CNV in the ocular fundus of the monkeys having received theintravenous injection of ICG at a dose of 0.5 mg/kg to 1 mg/kg, arerespectively able to predict and indicate the time-dependent changes inthe concentrations of NPe6 as distributed and remaining in the vascularwalls of the retinal normal vascular vessels and the time-dependentchange in the concentration of NPe6 as distributed and remaining in thevascular walls of CNV in the ocular fundus of the test monkeys havingreceived the intravenous injection of NPe6 at a dose of 0.5 mg/kg to 1mg/kg.

Consequently, the present inventors have now devised a new processwherein ICG is, at first, intravenously injected at a dose of 0.5 mg/kgto 1 mg/kg to the test monkeys prior to an intravenous injection ofNPe6, and wherein the known diagnosis method of making the infraredfluorescence fundus angiography of the ocular fundus is utilized inorder to estimate either such a time point at which the intensity of theinfrared fluorescence emitted from the ICG having distributed,accumulated and remaining in the vascular walls of the retinal normalvascular vessels is decreased to a significantly lower value than thatof the intensity of the infrared fluorescence emitted from the ICGhaving distributed, accumulated and remaining in the vascular walls ofCNV in the ocular fundus, or such a time point at which the infraredfluorescence of ICG has been eliminated from the vascular walls of theretinal normal vascular vessels, whereas the infrared fluorescence ofICG having accumulated and remaining in the vascular walls of CNV in theocular fundus is still observable and appreciable at a significantintensity, and wherein calculation is then made of a value of such atime-gap as extended between the time point estimated in the above andthe first time of the intravenous injection of ICG.

The present inventors have thus now presumed that it is made possible bythe so devised process as above to estimate and decide such anappropriate time-point at which the irradiation of a laser light of 664nm-wavelength is to be started for the photo-excitation of NPe6 asinjected, and namely at which the concentration of the NPe6 havingdistributed and remaining in the vascular walls of the retinal normalvascular vessels of the test monkeys having received the intravenousinjection of NPe6 has decreased to a significantly lower value than thatof the concentration of the NPe6 having accumulated and remaining in thevascular walls of CNV in the ocular fundus of the monkeys. On the basisof these findings of the present inventors, a second aspect of thisinvention as described hereinafter has now been accomplished.

In a second aspect of this invention, therefore, there is provided aphotodynamic therapy method for occluding selectively such choroidalneovascular vessels and/or retinal neovascular vessels as formed in amammalian animal having an ocular fundus tissue comprising the retinalnormal parenchymal tissue, the retinal normal vascular vessels and thechoroidal normal vascular vessels lying under the retina, as well aschoroidal neovascular vessels and/or retinal neovascular vessels, bydeveloping actions of the photochemical reaction of an administeredphotosensitizer as excited with an irradiating laser light, but withoutinvolving a substantial damage or impairment in the retinal normalparenchymal tissue, the retinal normal vascular vessels and thechoroidal normal vascular vessels, characterized in that the methodcomprises:

-   (a) administering firstly indocyanine green intravenously to the    animal at a dose of 0.5 mg/kg to 1 mg/kg at a vein of said animal;-   (b) allowing that the indocyanine green, which is carried along by    the blood streams circulating in the ocular retinal central artery    and ciliary artery, is uptaken in the endothelial cell layers of the    retinal normal vascular vessels and also in the endothelial cell    layers of the choroidal normal vascular vessels as well as in the    endothelial cell layers of the choroidal neovascular vessels and/or    the endothelial cell layers of the retinal neovascular vessels, and    allowing that the indocyanine green is then distributed and    accumulated in and being eliminated or cleared from the vascular    walls of said normal vascular vessels and said neovascular vessels;-   (c) subsequently irradiating and scanning a laser light containing a    light of a 790 nm-wavelength at a sufficient fluence of said laser    light, intermittently or continuously, through the surface of ocular    cornea, pupil, lens and vitreous fluid of the eye, to and on the    ocular fundus tissue of the animal, so as to induce emission of an    infrared fluorescence from the photo-excited indocyanine green    having distributed in the vascular walls of said normal vascular    vessels and said neovascular vessels in said ocular fundus tissue;    and further observing intermittently or continuously the intensity    of the infrared fluorescence which is emitted from the indocyanine    green having been exposed to the laser light of 790 nm-wavelength    and having distributed in the vascular walls of the retinal normal    vascular vessels as well as in the vascular walls of the choroidal    neovascular vessels and/or the vascular walls of the retinal    neovascular vessels, with using a fluorescence ocular fundus    angiography apparatus or a fluorescence microscope for funduscopy,    for this observation;-   (d) using said fluorescence ocular fundus angiography apparatus or    fluorescence microscope for funduscopy during the irradiation of the    790 nm-laser light, to estimate such a time point at which, in the    course of the elimination or clearance of indocyanine green out of    the vascular walls of the retinal normal vascular vessels and the    choroidal normal vascular vessels, the intensity of the infrared    fluorescence emitted from the indocyanine green present in the    vascular walls of the retinal normal vascular vessels under    observation can be observed to have decreased to a lowered value of    about one-half-folds or less, particularly ⅓-folds or less of the    intensity of the infrared fluorescence emitted from the indocyanine    green remaining in the vascular walls of the neovascular vessels    under observation; and thus to estimate such time point at which it    can be revealed from the observation of the decreases in the    intensity of the infrared fluorescence emitted from indocyanine    green by means of said fluorescence ocular fundus angiography    apparatus or fluorescence microscope for funduscopy that the    concentration of the indocyanine green present in the vascular walls    of the retinal normal vascular vessels under observation has    decreased to a lowered value of about one-half-folds or less,    particularly ⅓-folds or less of the concentration of the indocyanine    green remaining in the vascular walls of the neovascular vessels    under observation;-   (e) calculating in a “minute” or “second” unit, such a time-gap as    extended between the time of the first intravenous injection of    indocyanine green and the aforesaid time point as estimated in the    above step (d), namely the time point as estimated in the above    step (d) at which the intensity of the infrared fluorescence emitted    from the indocyanine green present in the vascular walls of the    retinal normal vascular vessels has decreased to the lowered value    of about one-half-folds or less of the intensity of the indocyanine    green remaining in the vascular walls of the neovascular vessels as    prescribed in the above step (d);-   (f) allowing a time to pass by the time when it can observed that    the infrared fluorescence of the indocyanine green remaining in the    vascular walls of the choroidal neovascular vessels and/or retinal    neovascular vessels has disappeared completely;-   (g) administering then mono-L-aspartyl chlorin e6 or a    pharmaceutically acceptable salt thereof, particularly    mono-L-aspartyl chlorin e6 tetra-sodium salt to the animal    intravenously at a dose in a range of 0.5 mg/kg to 10 mg/kg,    preferably 0.5 mg/kg to 1 mg/kg (as calculated on the weight basis    of NPe6) as the photosensitizer at a vein of said animal, after the    time when the infrared fluorescence of the indocyanine green    remaining in the vascular walls of said neovascular vessels under    the irradiation of the 790 nm-wavelength laser light can be observed    to have disappeared completely as described in the above step (f);-   (h) allowing that mono-L-aspartyl chlorin e6 or a salt thereof,    particularly mono-L-aspartyl chlorin e6 tetra-sodium salt as the    photosensitizer, which is carried along by the blood streams    circulating in the ocular retinal central artery and ciliary artery,    is uptaken in the endothelial cell layers of the retinal normal    vascular vessels and also in the endothelial cell layers of the    choroidal normal vascular vessels as well as in the endothelial cell    layers of the choroidal neovascular vessels and/or the endothelial    cell layers of the retinal neovascular vessels, and allowing that    the administered chlorin e6 compound is then distributed and    accumulated in and being eliminated or cleared from the vascular    walls of said normal vascular vessels and of said neovascular    vessels;-   (i) permitting the administered photosensitizer, mono-L-aspartyl    chlorin e6 compound, to be accumulated and remain in the vascular    walls of the choroidal neovascular vessels and/or the retinal    neovascular vessels, while the chlorin e6 compound is concurrently    eliminated or cleared out of the vascular walls of the normal    vascular vessels of the retina and choroid;-   (j) at such time point when a time duration, which has a time length    equal to that of the said time-gap (in the “minute” or “second”    unit) as calculated in the above step (e), has just passed after the    time of the intravenous injection of the mono-L-aspartyl chlorin e6    substance; starting to irradiate a laser light of 664 nm-wavelength    in the form of a thin laser beam, via the surface of cornea, pupil    and vitreous fluid of the eye, exclusively to such targeted lesions    comprising the neovasculature formed of said neovascular vessels in    the ocular fundus, in such a way that said laser light of 664    nm-wavelength is irradiated at a fluence of said laser light    necessary to excite the photosensitizer remaining in the vascular    walls of the neovascular vessels; and-   (k) subsequently permitting that the lumens of the neovascular    vessels contained in said lesions as irradiated with the laser light    of 664 nm-wavelength are occluded by the developed actions of the    photochemical reaction of the laser-excited photosensitizer    remaining in the vascular walls of said neovascular vessels, whereby    a selective occlusion of the choroidal neovascular vessels and/or    the retinal neovascular vessels is achieved.

In the method of the second aspect of this invention, it is possiblethat, in the step (j) of the method, the irradiation of the laser lightof 664 nm-wavelength via the surface of cornea, pupil and vitreous fluidof the eye exclusively to the targeted lesions comprising theneovasculatures formed of the neovascular vessels in the ocular fundusis started at such a time point when a time period of 10 to 70 minutes,preferably 20 to 70 minutes has lapsed after the time of the intravenousadministration of mono-L-aspartyl chlorin e6 or a salt thereof,particularly mono-L-aspartyl chlorin e6 tetra-sodium salt; provided thatsaid time point is the such time point when there has just passed a timeduration which has a time length equal to that of the said time-gap ascalculated in the step (e) of the method according to the second aspectof this invention.

In the method of the second aspect of this invention, it is preferredthat the laser light of 664 nm-wavelength, which is irradiated to thetargeted lesions comprising the neovascular vessels in the ocularfundus, has an irradiance of 10 to 1500 mW/cm², preferably 0.5 to 0.8W/cm² on the top retina face, as measured at the corneal surface bymeans of an optical power meter, and that the laser light of 664nm-wavelength is irradiated for a duration of 10 to 300 second, and at afluence of the laser light in a range of 7.0 J/cm² to 250 J/cm²,preferably 7.5 J/cm² to 205 J/cm²; provided that said fluence of thelaser light is evaluated by multiplying the laser irradiance (in W/cm²)by the duration of the laser irradiation (in seconds), whereupon thelower is the dose of mono-L-aspartyl chlorin e6, the fluence of thelaser light of 664 nm-wavelength to be irradiated is controlled to bethe higher, so as to achieve the selective occlusion of the neovascularvessels present in the target lesions.

Besides, in the method of the second aspect of this invention, it ispossible that the irradiation of the laser light of 664 nm-wavelength isstarted in the step (j) of the method at such a time point when a timeperiod of 20 to 70 minutes has lapsed after the time of the intravenousadministration of mono-L-aspartyl chlorin e6 or a salt thereof,particularly mono-L-aspartyl chlorin e6 tetra-sodium salt, as long asthe chlorin e6 compound is given at a dose of 0.5 mg/kg to 10.0 mg/kg(as calculated on the weight basis of NPe6), whereupon the fluence ofthe laser light of 664 nm-wavelength to be irradiated is controlledwithin a range of 7 J/cm² to 205 J/cm².

In the second aspect method, it is also possible that the irradiation ofthe laser light of 664 nm-wavelength is started in the step (j) of themethod at such a time point when a time period of 20 to 30 minutes haslapsed after the time of the intravenous administration ofmono-L-aspartyl chlorin e6 or a salt thereof, particularlymono-L-aspartyl chlorin e6 tetra-sodium salt, as long as the chlorin e6compound is given at a dose of 0.5 mg/kg to 0.9 mg/kg (as calculated onthe weight basis of NPe6), whereupon the fluence of the laser light of664 nm-wavelength to be irradiated is controlled within a range of 175J/cm² to 205 J/cm².

In the method of the second aspect of this invention, it is furthermorepossible that the irradiation of the laser light of 664 nm-wavelength isstarted in the step (j) of the method at such a time point when a timeperiod of 30 to 60 minutes has lapsed after the intravenousadministration of mono-L-aspartyl chlorin e6 or a salt thereof,particularly mono-L-aspartyl chlorin e6 tetra-sodium salt, as long asthe chlorin e6 compound is given at a dose of 1 mg/kg to 1.9 mg/kg (ascalculated on the weight basis of NPe6), whereupon the fluence of thelaser light of 664 nm-wavelength to be irradiated is controlled within arange of 30 J/cm² to 175 J/cm², preferably 34 J/cm² to 171 J/cm².

In the second aspect method of this invention, it is further possiblethat the irradiation of the laser light of 664 nm-wavelength is startedin the step (j) of the method at such a time point when a time period of60 minutes has lapsed after the time of the intravenous administrationof mono-L-aspartyl chlorin e6 or a salt thereof, particularlymono-L-aspartyl chlorin e6 tetra-sodium salt, as long as the chlorin e6compound is given at a dose of 2 mg/kg to 9.5 mg/kg (as calculated onthe weight basis of NPe6), whereupon the fluence of the laser light of664 nm-wavelength to be irradiated is controlled within a range of 30J/cm² to 45 J/cm².

In the method of the second aspect of this invention, it is moreoverpossible that the irradiation of the laser light of 664 nm-wavelength isstarted in the step (j) of the method at such a time point when a timeperiod of 60 to 70 minutes has lapsed after the time of the intravenousadministration of mono-L-aspartyl chlorin e6 or a salt thereof,particularly mono-L-aspartyl chlorin e6 tetra-sodium salt, as long asthe chlorin e6 compound is given at a dose of 9.5 mg/kg to 10 mg/kg(calculated on the weight basis of NPe6), whereupon the fluence of thelaser light of 664 nm-wavelength to be irradiated is controlled within arange of 7 J/cm² to 9 J/cm², preferably 7.5 J/cm² to 8 J/cm².

Also in the method of the second aspect of this invention, the mammaliananimal to be treated is a human having suffered from age-related maculardegeneration with the choroidal neovascular vessels, or a human havingsuffered from proliferative diabetic retinitis with proliferativeneovascular vessels.

As described hereinbefore in respect of the method of the second aspectof this invention, it has been revealed that, as long as ICG has beenintravenously injected at a dose of 0.5 mg/kg to 1 mg/kg to the testmonkeys having the experimental CNV in their ocular fundus, the patternof the time-dependent change in the intensity of the infraredfluorescence emitted from the ICG having been uptaken, distributed andremaining in the vascular walls of the retinal normal vascular vessels,as well as the pattern of the time-dependent change in the intensity ofthe infrared fluorescence emitted from the ICG having been uptaken,distributed and remaining in the vascular walls of CNV in the ocularfundus of the monkeys under test are, respectively, able to predict andindicate the pattern of the time-dependent change in the concentrationof the NPe6 having been uptaken, distributed and remaining in thevascular walls of the retinal normal vascular vessels, as well as thepattern of the time-dependent change in the concentration of the NPe6having been uptaken, distributed and remaining in the vascular walls ofCNV in the ocular fundus of the test monkeys in which NPe6 has beeninjected intravenously at dose of 0.5 mg/kg to 1 mg/kg.

Accordingly, the present inventors conclusively consider that, when ICGis intravenously injected at a dose of 0.5 mg/kg to 1 mg/kg to the testmonkeys having the experimental CNV in their ocular fundus and then NPe6is intravenously injected to said monkeys at a dose of 0.5 mg/kg to 10mg/kg, or desirably at a dose of 0.5 mg/kg to 1 mg/kg as same as thedose of the intravenously injected ICG, either at the same time as thetime of the intravenous injection of ICG, or immediately before orimmediately after the intravenous injection of ICG, it occurs that bothof ICG and NPe6 can concurrently be uptaken into and distributed in thevascular walls of the retinal normal vascular vessels and also in thevascular walls of the CNV in the ocular fundus of the monkeys. Thepresent inventors also consider conclusively that the ICG and NPe6,which have been intravenously injected at the same time or at asubstantially same time, are able to bring about the time-dependentchange in the concentration of ICG and also the time-dependent change inthe concentration of NPe6 within both of the vascular walls of theretinal normal vascular vessels and the vascular walls of the CNV in theocular fundus of the monkeys, so that the time-dependent change in theconcentration of NPe6 can take place concurrently in the same pattern ormanner as the time-dependent change in the concentration of ICG in saidvascular walls of the retinal normal vascular vessels and of the CNV.

Consequently, the present inventors have now attained such a conceptthat, when ICG is intravenously injected at a dose of 0.5 mg/kg to 1mg/kg, and then NPe6 is intravenously injected at such a dose of NPe6 assame as the dose of the intravenously injected ICG and either at thesame time as the time of the intravenous injection of ICG or at asubstantially same time as the time of the intravenous injection of ICG,it are practically feasible to conduct an indirect observation of thetime-dependent change in the concentration of the NPe6 havingdistributed and remaining in the vascular walls of the retinal normalvascular vessels, and also to conduct an indirect observation of thetime-dependent change in the concentration of the NPe6 havingdistributed and remaining in the vascular walls of the CNV in the ocularfundus, by carrying out such known diagnosis method of the infraredfluorescence fundus angiography for the ocular fundus by which directobservation can be made of the time-dependent change in the intensity ofthe infrared fluorescence of the ICG having distributed and remaining inthe vascular walls of the retinal normal vascular vessels, and by whichalso direct observation can be made of the time-dependent change in theintensity of the infrared fluorescence of the ICG having distributed andremaining in the vascular walls of the CNV in the ocular fundus.Further, the present inventors have now concluded that theabove-mentioned method of conducting the indirect observation of thetime-dependent changes in the intensity of ICG remaining in the vascularwalls of the retinal normal vascular vessels, as well as the indirectobservation of the time-dependent change in the intensity of ICGremaining in the vascular walls of the CNV in the ocular fundus, is ableto estimate and determine the appropriate time point at which there maybe started the required irradiation of the laser light of 664nm-wavelength to the neovasculature lesions formed of CNV in the ocularfundus. On the basis of these findings, a third aspect of this inventionhas been accomplished.

In the third aspect of this invention, therefore, there is provided aphotodynamic therapy method for occluding selectively such choroidalneovascular vessels and/or retinal neovascular vessels as formed in amammalian animal having an ocular fundus tissue comprising the retinalnormal parenchymal tissue, the retinal normal vascular vessels and thechoroidal normal vascular vessels lying under the retina, as well aschoroidal neovascular vessels and/or retinal neovascular vessels, bydeveloping actions of the photochemical reaction of an administeredphotosensitizer as excited with an irradiating laser light, but withoutinvolving any substantial damage or impairment in the retinal normalparenchymal tissue, the retinal normal vascular vessels and thechoroidal normal vascular vessels, characterized in that the methodcomprises:

-   (a) administering intravenously mono-L-aspartyl chlorin e6 or a    pharmaceutically acceptable salt thereof, particularly    mono-L-aspartyl chlorin e6 tetra-sodium salt as the photosensitizer    to the animal at a dose of 0.5 mg/kg to 10 mg/kg (as calculated on    the weight basis of NPe6) at a vein of the animal; and also    injecting intravenously indocyanine green to the animal at a dose in    a range of 0.5 mg/kg to 1 mg/kg, at the same time as or immediately    before or after said intravenous administration of the    mono-L-aspartyl chlorin e6 compound;-   (b) allowing that mono-L-aspartyl chlorin e6 or a salt thereof,    particularly mono-L-aspartyl chlorin e6 tetra-sodium salt,    administered as the photosensitizer and also indocyanine green,    which are carried along by the blood streams circulating in the    ocular retinal central artery and ciliary artery, are uptaken in the    endothelial cell layers of the retinal normal vascular vessels and    also in the endothelial cell layers of the choroidal normal vascular    vessels as well as in the endothelial cell layers of the retinal    neovascular vessels and/or the endothelial cell layers of the    choroidal neovascular vessels, and allowing that the chlorin e6    compound and indocyanine green as administered are then distributed    and accumulated in and being eliminated or clearedfrom the vascular    walls of said normal vascular vessels and said neovascular vessels;-   (c) subsequently irradiating and scanning a laser light containing a    light of 790 nm-wavelength, intermittently or continuously, through    the surface of ocular cornea, pupil, lens and vitreous fluid of the    eye, to and on the ocular fundus tissue of the animal, so as to    induce emission of an infrared fluorescence from the photo-excited    indocyanine green selectively, in the co-presences of the    mono-L-aspartyl chlorin e6 and indocyanine green having distributed    and remaining in the vascular walls of said vascular vessels of the    ocular fundus tissue; and further observing intermittently or    continuously, the intensity of the infrared fluorescence as emitted    from the indocyanine green having distributed and remaining in the    vascular walls of the retinal normal vascular vessels, as well as in    the vascular walls of the choroidal neovascular vessels and/or the    vascular walls of the retinal neovascular vessels, with using a    fluorescence ocular fundus angiography apparatus or fluorescence    microscope for funduscopy for this observation;-   (d) using said fluorescence ocular fundus angiography apparatus or    fluorescence microscope for funduscopy during the irradiation of 790    nm-wavelength, to estimate such a time point at which, in the course    of the eliminations or clearances of the mono-L-aspartyl chlorin e6    substance and indocyanine green from the vascular walls of the    retinal normal vascular vessels and the choroidal normal vascular    vessels, the intensity of the infrared fluorescence emitted from the    indocyanine green present in the vascular walls of the retinal    normal vascular vessels under observation can be observed to have    decreased to a lowered value of about one-half-folds or less,    particularly ⅓-folds or less of the intensity of the infrared    fluorescence emitted from the indocyanine green remaining in the    vascular walls of the neovascular vessels under observation; and    thus to estimate the such time point at which it can be revealed    from the observation of the decreases in the intensity of the    infrared fluorescence emitted from the indocyanine green by means of    the fluorescence ocular fundus angiography apparatus or fluorescence    microscope for funduscopy that the concentration of the    photosensitive mono-L-aspartyl chlorin e6 compound present in the    vascular walls of the retinal normal vascular vessels under    observation has decreased to a lowered value of about one-half-folds    or less, particularly ⅓-folds or less of the concentration of the    photosensitive mono-L-aspartyl chlorin e6 compound remaining in the    vascular walls of the neovascular vessels under observation;-   (e) at the time point so estimated in the above step (d) or within a    time of 1–10 minutes from the so estimated time point; starting to    irradiate a laser light of 664 nm-wavelength in the form of a thin    laser beam, via the surface of cornea, pupil and vitreous fluid of    the eye, exclusively to such targeted lesions comprising the    neovasculature formed of said neovascular vessels in the ocular    fundus, in such a way that said laser light of 664 nm-wavelength is    irradiated at a fluence of said laser light necessary to excite said    photosensitive chlorin e6 compound remaining in the vascular walls    of the neovascular vessels; and-   (f) subsequently permitting that the lumens of the neovascular    vessels contained in said lesions as irradiated with the laser light    of 664 nm-wavelength are occluded by the developed actions of the    photochemical reaction of the laser-excited mono-L-aspartyl chlorin    e6 compound remaining in the vascular walls of said neovascular    vessels, whereby a selective occlusion of the choroidal neovascular    vessels and/or the retinal neovascular vessels is achieved.

In the method of the third aspect of this invention, it is possiblethat, in the step (e) of this method, the irradiation of the laser lightof 664 nm-wavelength via the surface of cornea, pupil and vitreous fluidof the eye exclusively to the targeted lesions comprising theneovasculature formed of the neovascular vessels in the ocular fundus isstarted at such a time point when a time period of 10 to 70 minutes haslapsed after the time of the intravenous administration ofmono-L-aspartyl chlorin e6 or a salt thereof, particularlymono-L-aspartyl chlorin e6 tetra-sodium salt; provided that said timepoint is also the such time point when it can be revealed and estimatedfrom the observation of the decreases in the intensity of the infraredfluorescence as emitted from the indocyanine green having distributedand remaining in the vascular walls by excitation of the indocyaninegreen under the irradiation of the laser light containing the laserlight of 790 nm-wavelength, with using the fluorescence ocular fundusangiography apparatus or the fluorescence microscope for funduscopy,that the concentration of the photosensitive mono-L-aspartyl chlorin e6compound present in the vascular walls of the retinal normal vascularvessels has decreased to a lowered value of about one-half-folds or lessof the concentration of the photosensitive mono-L-aspartyl chlorin e6compound remaining in the vascular walls of the choroidal neovascularvessels and/or the retinal neovascular vessels.

In the method of the third aspect of this invention, it is also possiblethat, in the step (d) of this method, there is estimated such a timepoint when a time period of 20 to 70 minutes has lapsed after the timeof the intravenous injection of mono-L-aspartyl chlorin e6 or a saltthereof, particularly mono-L-aspartyl chlorin e6 tetra-sodium salt;provided that said time point is also such time point when it can berevealed from the observation with the fluorescence ocular fundusangiography apparatus or fluorescence microscope for funduscopy that theintensity of the infrared fluorescence emitted from the indocyaninegreen remaining in the vascular walls of the choroidal neovascularvessels and/or the retinal neovascular vessels has reached its peakvalue under the irradiation of the laser light containing the laserlight of 790 nm-wavelength; and provided that said time point is furtherthe such time point when it is also observed by said fluorescence ocularfundus angiography apparatus or fluorescence microscope for funduscopythat the infrared fluorescence as emitted from the indocyanine greenstill remaining in the vascular walls of the retinal normal vascularvessels has disappeared completely even under the irradiation of saidlaser light containing the light of 790 nm-wavelength; and it is alsopossible that in this method, subsequently just at the time point soestimated in the above, the laser light of 664 nm-wavelength is thenstarted in the step (e) of this method to be irradiated through thesurface of cornea, pupil and vitreous fluid of the eye exclusively tosuch targeted lesions comprising the neovasculature formed of theneovascular vessels in the ocular fundus.

In the method of the third aspect of this invention, it is preferredthat the laser light of 664 nm-wavelength which is irradiated to thetargeted lesions comprising the neovascular vessels in the ocularfundus, has an irradiance of 10 to 1500 mW/cm², preferably 0.5 to 0.8W/cm² on the top retina face, as measured at the corneal surface bymeans of an optical power meter, and said laser light of 664nm-wavelength is irradiated for a duration of 10 to 300 seconds and at afluence of the laser light in a range of 7.0 J/cm² to 250 J/cm²,preferably 7.5 J/cm² to 205 J/cm², provided that said fluence of thelaser light is evaluated by multiplying the laser irradiance (in W/cm²)by the duration of the laser irradiation (in seconds), whereupon thelower is then the dose of mono-L-aspartyl chlorin e6, the fluence of thelaser light of 664 nm to be irradiated is controlled to be the higher,so as to achieve the selective occlusion of the neovascular vesselspresent in the targeted lesions.

In the method of the third aspect of this invention, it is possible thatthe laser light of 664 nm-wavelength is started in the step (e) of thismethod to be irradiated at such a time point when a time period of 20 to70 minutes has lapsed after the time of the intravenous administrationof mono-L-aspartyl chlorin e6 or a salt thereof, particularlymono-L-aspartyl chlorin e6 tetra-sodium salt, as long as the chlorin e6compound is given at a dose of 0.5 mg/kg to 10.0 mg/kg (as calculated onthe weight basis of NPe6), and that the fluence of the laser light of664 nm-wavelength to be irradiated is controlled within a range of 7J/cm² to 205 J/cm².

In this method, it is also possible that the laser light of 664nm-wavelength is started in the step (e) of this method to be irradiatedat such a time point when a time period of 20 to 30 minutes has lapsedafter the time of the intravenous administration of mono-L-aspartylchlorin e6 or a salt thereof, particularly mono-L-aspartyl chlorin e6tetra-sodium salt, as long as the chlorin e6 compound is given at a doseof 0.5 mg/kg to 0.9 mg/kg (as calculated on the weight basis of NPe6),and that the fluence of the laser light of 664 nm-wavelength to beirradiated is controlled within a range of 175 J/cm² to 205 J/cm².

In the method of the third aspect of this invention, it is furthermorepossible that the laser light of 664 nm-wavelength is started in thestep (e) of this method to be irradiated at such a time point when atime period of 30 to 60 minutes has lapsed after the time of theintravenous administration of mono-L-aspartyl chlorin e6 or a saltthereof, particularly mono-L-aspartyl chlorin e6 tetra-sodium salt, aslong as the chlorin e6 compound is given at a dose of 1 mg/kg to 1.9mg/kg (as calculated on the weight basis of NPe6), and that the fluenceof the laser light of 664 nm-wavelength to be irradiated is controlledwithin a range of 30 J/cm² to 175 J/cm², preferably 34 J/cm² to 171J/cm².

In this method, it is moreover possible that the laser light of 664nm-wavelength is started in the step (e) of this method to be irradiatedat such a time point when a time period of 60 minutes has lapsed afterthe time of the intravenous administration of mono-L-aspartyl chlorin e6or a salt thereof, particularly mono-L-aspartyl chlorin e6 tetra-sodiumsalt, as long as the chlorin e6 compound is given at a dose of 2 mg/kgto 9.5 mg/kg (as calculated on the weight basis of NPe6), and that thefluence of the laser light of 664 nm-wavelength to be irradiated iscontrolled within a range of 30 J/cm² to 45 J/cm².

In the method of the third aspect of this invention, it is also feasiblethat the laser light of 664 nm-wavelength is started in the step (e) ofthis method to be irradiated at such a time point when a time period of60 to 70 minutes has lapsed after the time of the intravenousadministration of mono-L-aspartyl chlorin e6 or a salt thereof,particularly mono-L-aspartyl chlorin e6 tetra-sodium salt, as long asthe chlorin e6 compound is given at a dose of 9.5 mg/kg to 10 mg/kg (ascalculated on the weight basis of NPe6), and that the fluence of thelaser light of 664 nm-wavelength to be irradiated is controlled within arange of 7 J/cm² to 9 J/cm², preferably 7.5 J/cm² to 8 J/cm².

In the method of the third aspect of this invention, the mammaliananimal to be treated may be a human having suffered from age-relatedmacular degeneration with the choroidal neovascular vessels, or a humanhaving suffered from proliferative diabetic retinitis with proliferativeneovascular vessels in the retina.

In the methods of this invention as described hereinbefore, it ispreferred to use mono-L-aspartyl chlorin e6 tetra-sodium salt as thephotosensitizer. Mono-L-aspartyl chlorin e6 tetra-sodium salt(abbreviated as NPe6) may be intravenously administered in the form ofits aqueous solution. In the aqueous solution of NPe6 for theintravenous injections, generally, the solvent usable for the injectionsmay be, for example, water, aqueous ethanol or an aqueous polyol (forexample, glycerol, propylene glycol, a liquid polyethylene glycol, andothers), or a desirable mixture of two or more of these solvents. Thefluidity of the injectable aqueous solution may be adjusted byincorporation of a viscosity adjustor such as lecithin. In many cases,an isotonic agent such as sugar or sodium chloride may preferably beincorporated therein.

In a fourth aspect of this invention, there is provided as a novelproduct an intravenously administrable pharmaceutical composition foruse in a diagnosis or a selective occlusion of choroidal neovascularvessels and/or retinal neovascular vessels in the ocular fundus of eyeaccording to a photodynamic therapy method, characterized in that saidpharmaceutical composition is in the form of a single dosage unit forthe intravenous injection, and that the composition containsmono-L-aspartyl chlorin e6 tetra-sodium salt in a proportion thereofcorresponding to a dose of 0.5 mg/kg to 10 mg/kg, preferably a dose of0.5 mg/kg to 1 mg/kg of mono-L-aspartyl chlorin e6 tetra-sodium salt asan effective component, and that the composition further containsindocyanine green in a proportion thereof corresponding to a dose of 0.5mg/kg to 1 mg/kg of indocyanine green as an indicator capable ofdetecting the time-dependent decrease of the concentration of theadministered mono-L-aspartyl chlorin e6 tetra-sodium salt havingaccumulated and remaining in the vascular walls of the ocular, choroidalneovascular vessels and/or retinal neovascular vessels after theintravenous administration of said composition was done, and that themono-L-aspartyl chlorin e6 tetra-sodium salt and indocyanine greencontained in the intravenously administered single dosage unit of saidcomposition are dissolved in an aqueous carrier for the intravenousinjection.

The pharmaceutical composition according to the fourth aspect of thisinvention may suitably be used in the above-mentioned method of thethird aspect of this invention. In said pharmaceutical composition, itis preferred that the weight ratio of mono-L-aspartyl chlorin e6tetra-sodium salt to indocyanine green as contained in the single dosageunit of the composition is in a range of 1:2 to 1:0.05, or moredesirably in a range of 2:1 to 1:2, or is at a weight ratio which isnear to the ratio of 2:1 to 1:2.

A further aspect of this invention includes a use of mono-L-aspartylchlorin e6 tetra-sodium salt in the manufacture of an intravenouslyadministrable pharmaceutical composition for use in a diagnosis or aselective occlusion of ocular, choroidal neovascular vessels and/orretinal neovascular vessels of eye according to a photodynamic therapymethod, wherein said pharmaceutical composition is in the form of asingle dosage unit for the intravenous injection, and wherein thecomposition contains mono-L-aspartyl chlorin e6 tetra-sodium salt in aproportion thereof corresponding to a dose of 0.5 mg/kg to 10 mg/kg,preferably a dose of 0.5 mg/kg to 1 mg/kg of mono-L-aspartyl chlorin e6tetra-sodium salt as an effective component, and wherein the compositionfurther contains indocyanine green in a proportion thereof correspondingto a dose of 0.5 mg/kg to 1 mg/kg of indocyanine green as an indicatorcapable of detecting the time-dependent decrease of the concentration ofthe administered mono-L-aspartyl chlorin e6 tetra-sodium salt havingaccumulated and remaining in the vascular walls of the ocular, choroidalneovascular vessels and/or retinal neovascular vessels after theintravenous administration of said composition was done.

BEST MODE FOR CARRYING OUT THE INVENTION

This invention is now illustrated in detail with reference to thefollowing Test Examples and illustrative Examples, but the invention isnot limited to these Examples.

TEST EXAMPLE 1 Referential Example 1

In this Example, experiments were conducted to examine that, when theintravenous administration of mono-L-aspartyl chlorin e6 tetra-sodiumsalt had been done to monkeys having the experimentally producedchoroidal neovaucular vessels (CNV) in the ocular fundus of the monkeys,the concentration of the mono-L-aspartyl chlorin e6 tetra-sodium salthaving been uptaken, distributed and remaining in the vascular walls ofthe retinal normal vascular vessels of the monkeys, as well as theconcentration of the mono-L-aspartyl chlorin e6 tetra-sodium salt havingbeen uptaken, distributed and remaining in the vascular walls of thechoroidal neovascular vessels (CNV) of the monkeys were time-dependentlychanging in manners or patterns different from each other.

The time-dependent changes in the concentrations of the mono-L-aspartylchlorin e6 tetra-sodium salt (NPe6) having distributed and remaining inthe vascular walls of the retinal normal vascular vessels and also inthe vascular walls of the choroidal neovascular vessels (CNV) wereobserved by a fluorescence fundus angiography process, wherein thechanging intensities of the red fluorescence as emitted from NPe6present in said vascular walls were recorded by photography underirradiation of the laser light beam of 488 nm by means of asfluorescence fundus camera apparatus, and wherein the resultingphotographed images of different gradations were examined by visualobservation.

The detailed procedures of the above experiments are as follows. Thus, abeam of a laser light of 647 nm wavelenght as emitted from a kryptonlaser generator [Coherent Medical Laser generator, manufactured by NovuaOmni Co.; having an output of 500 to 900 mW] was irradiated as alaser-irradiating spot of a diameter of 50 μm onto the ocular fundi of 8mature monkeys of the genus Macaca (of body weights of 3.5 kg to 6.0 kg)for 0.1 seconds, according to a modification of the Ryan's method [Arch.Ophthalmol., vol 100; pp. 1804–1809 (1982)]. It is said that theresulting damage in the Bruch's membrane, which were incurred by theirradiation of the 647 nm laser light, is very effective for theincidence of ocular neovascular vessels. Thereby, there were induced theexperimental choroidal neovascular vessels (abbreviated as CNV). Theinduced CNV are the neovascular vessels as branched from the choroidalnormal vascular vessels in the ocular fundus. Such CNV have infiltratedin the subretinal space, namely the space between the sensory retina andchoroids, to involve the occurrence of exudation, bleeding and retinaldetachment. The formation of the neovasculature lesions comprising theCNV was confirmed by a process which comprised intravenously injectingfluorescein or indocyanine green to the so treated monkeys andsubsequently carrying out the diagnosis method according to afluorescence fundus angiography of the ocular fundus. It was thusconfirmed that a great number of the neovasculature lesions comprisingthe experimental CNV could be produced in the subretinal space.

-   (i) To the monkeys which had the neovasculature lesion comprising    the CNV so produced by the modified Ryan's method in the subretinal    space of the ocular fundus, was intravenously administered    mono-L-aspartyl chlorin e6 tetra-sodium salt (sometimes abbreviated    as NPe6 hereinafter) at a dose of 20 mg/kg at a vein in the lower    limbs. Then, using a scanning laser ophthalmoscope (abbreviated as    SLO; manufactured by Rodenstock, Co., Germany) which emits a beam of    argon laser light of a wavelength of 488 nm, a beam of 488 nm argon    laser light was continuously irradiated to the ocular fundus for 10    minutes, immediately after the time of the intravenous injection of    NPe6. By the excitation of NPe6 under irradiation of the 488 nm    laser light beam, the NPe6 having distributed in the fundus tissues    and in the vascular walls of the ocular fundus could emit a red    fluorescence.

In the course of said irradiation of 488 nm laser light beam for 10minutes, the images of the red fluorescence as emitted in the ocularfundus were recorded by photography on a video tape by means of ahigh-sensitivity television fundus camera apparatus. Thereafter, atintervals of 10 minutes, a beam of 488 nm laser light was repeatedlyirradiated to the ocular fundus each for about one minute. At each ofthe irradiations of the 488 nm laser light beam, the images of the redfluorescence emitted from the vascular vessels of the ocular fundus wererecorded on the video tape. At the time of 60 minutes after the time ofthe intravenous injection of NPe6, the irradiation of the 488 nm laserlight beam was terminated. The images of the fluorescence emitted fromthe ocular fundus vascular vessels were then reproduced from therecording video tape for the purpose of observation.

According to these observations, it was revealed that the redfluorescence of the NPe6 having distributed and remaining in thevascular walls of the retinal normal vascular vessels was appreciablyobservable at its prominent intensity, but the red fluorescence of theNPe6 having distributed and remaining in the vascular walls of thechoroidal neovascular vessels (CNV) was observable slightly butdetectable at its weak intensity, at the time of 7.5 minutes after thetime of the intravenous injection of NPe6.

It was also revealed that, at the time of 20 minutes after the time ofthe intravenous injection of NPe6, the red fluorescence emitted from theNPe6 present in the retinal normal vascular vessels had decreased to alower and hardly detectable intensity, but the red fluorescence emittedfrom the NPe6 present in the choroidal neovascular vessels (CNV) wasconfirmed to remain at a higher and prominently observable intensity.From these observations, it was revealed that NPe6 had accumulated andremained selectively in the vascular walls of the choroidal neovascularvessels still at said time of 20 minutes after the injection of NPe6.Accordingly, it was found that the NPe6, which was once distributed inthe vascular walls of the retinal normal vascular vessels after theintravenous injection of NPe6, could be substantially entirely clearedor eliminated from the vascular walls of the retinal normal vascularvessels already at the time of 20 minutes after the intravenousinjection of NPe6, and that the NPe6 as once distributed could beaccumulated and remain preferentially in the vascular walls of thechoroidal neovascular vessels (CNV), in contrast.

It was further found that, at the time of 60 minutes after theintravenous injection of NPe6, the red fluorescence emitted from theNPe6 present in the retinal normal vascular vessels was never observablewith any of the fluorescence images as reproduced from said recordingvideo tape, but the red fluorescence of the Npe6 present in thechoroidal neovascular vessels (CNV) could be observed at a high andprominent intensity. Consequently, it has been confirmed that the NPe6can accumulate and remain at a significant concentration preferentiallyin the vascular walls of the neovascular vessels (CNV) in the ocularfundus.

At the time of 65 to 70 minutes after the time of the intravenousinjection of NPe6, three eyeballs were enucleated from the monkey undertest, and each eyeball was cut into 4 pieces. These cut pieces of theeyeballs were frozen in a bath of liquid nitrogen. Cross-section sampleseach having a thickness of 5 μm were prepared from the ocular fundustissue region of the frozen pieces of the eyeballs. These cross-sectionsamples were placed under an excitation light of a wavelength of 380 to420 nm, and the red fluorescence emitted from the NPe6 remaining in saidsamples was observed under a fluorescence microscope. The redfluorescence was almost not observable in the cross-section samples asprepared from the retinal parenchymal region of the ocular fundus. Inthe cross-section samples containing the retinal normal vascularvessels, only a very slight degree of the red fluorescence was observedat the retinal normal vascular vessels present therein. In contrast, inthe cross-section samples containing the choroidal neovascular vessels(CNV), the red fluorescence emitted from the choroidal neovascularvessels was observed confirmably at a significant intensity.

Accordingly, it has been confirmed that, when NPe6 is intravenouslyadministered to the test monkeys at a dose of 20 mg/kg, the administeredNPe6 can accumulate and remain at a significant concentrationpreferentially in the vascular walls of the choroidal neovascularvessels, but a substantial major part of NPe6 can have been cleared oreliminated completely from the vascular walls of the retinal normalvascular vessels of the ocular fundus already at the time of 60 minutesafter the intravenous injectionof NPe6.

-   (ii) NPe6 was intravenously administered at a dose of 0.5 mg/kg or    1.0 mg/kg in the same way as described in the above (i), to the test    monkeys having the neovasculature lesions comprising the CNV as    produced by the above described method, within the retina of the    ocular fundus.

In the same way as described in the above (i), a beam of a laser lightof 488 nm-wavelength was irradiated by SLO at an enhanced fluence to theocular fundi of the test monkeys continuously for 10 minutes immediatelyafter the time of the intravenous injection of NPe6. At the time of 15minutes after the time of the intravenous injection of NPe6, theirradiation of the beam of 488 nm-laser light to the ocular fundi wasagain started and effected for a duration of 10 minutes. During this 10minutes-irradiation of the 488 nm-laser light to the ocular fundi, theimages of the red fluorescence emitted from the ocular fundi wererecorded by photography on a video tape by means of a high-sensitivitytelevision fundus camera.

The images as recorded of the red fluorescence were reproduced from thephotographs in the recording video tape and then were visually observed.According to these observations, it has been found that the redfluorescence emitted from the NPe6 present in the retinal normalvascular vessels has substantially entirely been eliminated anddisappeared, but the red fluorescence emitted from the NPe6 present inthe choroidal neovascular vessels can be confirmably observed at aslight but appreciable intensity at the time point of 20 minutes afterthe time of the intravenous injection of NPe6, in a case when NPe6 wasinjected at a dose of 0.5 mg/kg. Additionally, it has been found thatthe red fluorescence emitted from the NPe6 present in the retinal normalvascular vessels has substantially entirely been eliminated, but the redfluorescence emitted from the NPe6 present in the choroidal neovascularvessels can be confirmed at a significant and appreciable intensity atthe time point of 30 minutes after the time of the intravenous injectionof NPe6, in a case when NPe6 was injected at a dose of 1.0 mg/kg.

TEST EXAMPLE 2

This Example illustrates an embodiment of the method of the first aspectof this invention.

In the experiments of this Test Example 2, there were used 31neovasculature lesions chosen from among the neovasculature lesionswhich comprised the experimental choroidal neovascular vessels (CNV) asproduced in the ocular fundi of the monkeys of the genus Macaca by themodified Ryan's method set out in the Test Example 1 above.

A solution of mono-L-aspartyl chlorin e6 tetra-sodium salt (sometimesabbreviated as NPe6) dissolved in physiological saline at aconcentration of 5.0 mg/ml was intravenously administered at a vein inthe lower limbs of the test monkeys having the experimental CNV. Thedose of the administration of NPe6 was preset at 0.5 mg/kg, 1 mg/kg, 2mg/kg or 10 mg/kg.

At the time of 10 minutes after the time of the intravenous injection ofNPe6, the operation for irradiation and scanning of a beam of the laserlight of 488 nm-wavelength was started to be effected towards the ocularretina. During this irradiation of the 488 nm-laser light, the images ofthe red fluorescences emitted from the retinal normal vascular vesselsand from the choroidal neovascular vessels were observed by an apparatusfor the fluorescence ocular fundus angiography of the ocular fundus(namely, an infrared light fundus camera).

When NPe6 was intravenously injected at a dose of 0.5 mg/kg, it wasobserved that the red fluorescence of NPe6 had been substantiallyentirely eliminated from the retinal normal vascular vessels already atthe time point of 20 minutes after the time of the intravenous injectionof NPe6, but the red fluorescence of the NPe6 present in theneovasculature lesions comprising CNV was confirmed to continue to emitstill at said time point of 20 minutes. At said time of 20 minutes afterthe time of the injection of NPe6, irradiation of 664 nm-laser light asemitted from a semiconductor-type laser-generator [manufactured byMatsushita Industry System, Co., Ltd., Japan] was started to be effectedas a laser-irradiating spot of 1 mm diameter selectively to only one ofthe said lesions under test. The 664 nm-laser light was irradiated for300 seconds at a fluence of 204 J/cm². The details of the experimentalconditions for this irradiation of the 664 nm-laser light are shown inTable 1 given hereinafter.

When NPe6 was intravenously injected at a dose of 1 mg/kg, it wasobserved that the red fluorescence of the administered NPe6 present inthe retinal normal vascular vessels had substantially entirely beeneliminated but the red fluorescence of the NPe6 present in theneovasculature lesions was confirmed to continue to emit at the timepoint of 30 minutes after the time of the intravenous injection of NPe6.The observations of the red fluorescence of NPe6 were made by theapparatus for the fluorescence ocular fundus angiography of the ocularfundus. At said time point of 30 minutes after the NPe6 injection,irradiation of the 664 nm-laser light was started to be effected as alaser-irradiating spot of 1 mm diameter selectively to 3 lesions chosenfrom among the neovasculature lesions under test. The laser light wasirradiated for 60 to 300 seconds at different fluences. The details ofthe experimental conditions for these irradiations of the 664 nm-laserlight are shown in Table 1 below.

When NPe6 was intravenously injected at a dose of 2 mg/kg or 10 mg/kg,it was observed that the red fluorescence of the NPe6 present in theretinal normal vascular vessels had substantially entirely beeneliminated, but the red fluorescence of the NPe6 present in theneovasculature lesions was confirmed by said fluorescence ocular fundusangiography apparatus to continue to emit still at the time point of 60minutes after the time of the NPe6 injection. At said time of 60 minutesafter the time of the injection of NPe6, irradiation of the 664 nm-laserlight was started to be effected to the neovasculature lesions. At thedose of 2 mg/kg of NPe6, two neovasculature lesions were irradiated withthe 664 nm-laser light. At the dose of 10 mg/kg of NPe6, fourneovasculature lesions were irradiated with the 664 nm-laser light. The664 nm-laser light was irradiated at different fluences for 60 secondsor for 10 seconds. The details of the experimental conditions for theseirradiations of the 664 nm-laser light are shown in Table 1.

In the above experiments, the irradiations of the 664 nm-laser lightwere designed to be carried out under such experimental conditions thatthe laser light was irradiated at an irradiance of 0.57 to 0.75 W/cm²,for a duration of 10 to 300 seconds, and at a fluence of 7.5 to 204J/cm².

Among the 31 neovasculature lesions comprising CNV as employed in thetests, 13 neovasculature lesions were treated by the administration ofNPe6 but without irradiation of the 664 nm-laser light and were referredto as First Control group. While, 8 neovasculature lesions were treatedby the irradiation of the 664 nm-laser light but without administrationof NPe6 and were referred to as Second Control group.

After the treatment with the irradiation of 664 nm-laser light wasterminated, the test monkeys having the neovasculature lesions, eithertreated or untreated, were fed for 1 week under routine feedingconditions. After the feeding for one week, the effects of the abovetreatments were judged by a fluorescence fundus angiography withfluorescein and by a fluorescence fundus angiography with indocyaninegreen (ICG). For the fluorescein fundus angiography, the ocular fundi ofthe monkeys were observed and diagnosed by means of an ocular funduscamera apparatus made by Nikon Company, Japan, after the intravenousinjection of fluorescein. For the fluorescence fundus angiography withICG, the ocular fundi of the monkeys were observed and diagnosed bymeans of SLO or by means of an infrared light fundus camera apparatusfor the infrared ocular fundus angiography, after the intravenousinjection of ICG. Examinations were made of changes which have occurredin the treated neovasculature lesions and also in the retinalparenchymal tissue and in the retinal normal vascular vessels around theneovasculature lesions in the ocular fundus. In account of the soexamined changes occurred in the fundus, the therapeutic effects of thetreatments as conducted were judged. In the above experiments, it wasconfirmed that, in the totally 10 neovasculature lesions which weretreated by both of the administration of NPe6 and the irradiation of 664nm-laser light, the vascular lumens of the CNV present in said 10neovasculature lesions could be occluded without involving anysubstantial injury in the tissues present in the surrounding regionspositioned outside said 10 neovasculature lesions, and without involvingany substantial injury in the retinal normal vascular vessels. It isthus confirmed that the method of the first aspect of this invention isable to selectively occlude with success all the CNV present in theabove-mentioned 10 neovasculature lesions which have been treated with acombination of the administration of NPe6 and the irradiation of 664nm-laser light.

On the other hand, in the totally 21 neovasculature lesions of the FirstControl group and of the Second Control group, it was observed that anyof the CNV as treated could not be occluded.

Then, several speciemen fragments were excised out of the ocular fundustissues containing the regions of the neovasculature lesions comprisingthe CNV which had been selectively occluded by the method of thisinvention in the above experiments. These speciemen fragments of theocular fundus tissues were then pathologically examined under an opticalmicroscope. From the microscopic examination, it was shown that thevascular lumens of the choroidal neovascular vessels (CNV), which wereselectively occluded by the method of this invention, were filled andoccluded with cellular debris, and that the retinal normal vascularvessels and the choroidal normal vascular vessels which are positionedadjacently to the choroidal neovascular vessels (CNV) as selectivelyoccluded by the method of this invention, were remaining intact ornearly intact. Additionally, it was shown that the biological structureof the retinal inner layer, which is adjacent to the so treatedchoroidal neovascular vessels (CNV), remained unchanged.

An electron microscopic examination of the CNV which were selectivelyoccluded by the method of this invention in the above experiments, hasrevealed that the endothelial cells present in the vascular walls of theselectively occluded CNV had been severely damaged, resulting indeterioration of the normal structure of the endothelial cells. It wasalso revealed that the vascular lumens of the so selectively occludedCNV were embedded with the cytoplasmic debris and/or with bloodcell-deteriorated components. While, it was seen that the retinal normalvascular vessels and the choroidal normal vascular vessels which areadjacent to the selectively occluded CNV were not injured. Accordingly,it is assumed that the selective occlusion of CNV which is attainable inaccordance the method of this invention can be resulted due to theinjury as incurred in the endothelial cells of the vascular walls of theneovascular vessels of CNV.

The experimental conditions for the irradiation of the 664 nm-laserlight as effected in this Test Example 2, as well as the results of thePDT treatments for the selective occlusion of CNV are summarized inTable 1 below. In Table 1, NPe6 is the abbreviation of mono-L-aspartylchlorin e6 tetra-sodium salt.

TABLE 1 Time point for starting Number of CNV lesions irradiation of 664nm- Duration of containing the laser light, namely irradiation Fluenceof selectively occluded NPe6 lapse time (in minutes) Irradiance of of664 nm- 664 nm-laser Number of CNV after Test Run dose afteradministration 664 nm-laser laser light light CNV lesions irradiation ofNo. (mg/kg) of NPe6 light (W/cm²) (seconds) (J/cm²) tested 664 nm-laserlight 1 10 60 0.75 10 7.5 4 4 2 2 60 0.75 60 45 2 2 3 1 30 0.57 60 34.21 1 1 30 0.57 180 102.6 1 1 1 30 0.57 300 171 1 1 4 0.5 20 0.68 300 2041 1 First 10 — — — — 4 0 control 2 — — — — 2 0 group 1 — — — — 3 0 0.5 —— — — 4 0 Second 0 60 0.75 10 7.5 4 0 control 0 60 0.75 60 45 2 0 group0 60 0.75 300 225 2 0

TEST EXAMPLE 3 Referential Example 2

In this Example, experiments were carried out to examine and detectthat, when indocyanine green (ICG) [which is commonly used as aninfrared fluorescence agent in the infrared fluorescence fundusangiography for diagnosing the ocular fundus of patients withage-related macular degeneration] had been intravenously injected at adose of 0.5 to 1 mg/kg into the monkeys having the experimentalchoroidal neovascular vessels (CNV) in the ocular fundus of eye, theconcentration of ICG having uptaken, distributed and remaining in thevascular walls of the retinal normal vascular vessels, as well as theconcentration of ICG having uptaken, distributed and remaining in thevascular walls of the choroidal neovascular vessels (CNV) in the ocularfundus of the monkeys were time-dependently changing in differentmanners or patterns from each other.

In these experiments, the time-dependent changes in the concentrationsof ICG having distributed and remaining in the vascular walls of theretinal normal vascular vessels and in the vascular walls of thechoroidal neovascular vessels (CNV) in the ocular fundus of the monkeyswere examined by a process wherein a beam of laser light containing alight of 790 nm-wavelength (as emitted from a semiconductor type lasergenerator) was used as an excitation light, and wherein the changes inthe intensity of the infrared fluorescence emitted from the ICG asexcited within said vascular walls were continuously recorded byphotographs by means of an infrared light fundus camera apparatus (ofmodified TCR 50-IA type, manufactured by Topcon Co., Japan) for theinfrared fluorescence fundus angiography for diagnosing the ocularfundus, and wherein the photographed images of the fundus were observedvisually.

From these experiments, it has now been found that the manners orpatterns of the time-dependent changes in the concentrations of ICGhaving distributed and remaining in the vascular walls of the retinalnormal vascular vessels and in the vascular walls of the choroidalneovascular vessels (CNV) in the ocular fundus of the monkeys havingreceived the administration of ICG are always very much analogous to themanners or patterns in which the changes in the concentrations ofmono-L-aspartyl chlorin e6 tetra-sodium salt (NPe6) (as given at a doseof 0.5 to 10 mg/kg) having distributed and remaining in the vascularwalls of the retinal normal vascular vessels and in the vascular wallsof the choroidal neovascular vessels (CNV) of the monkeys took placetime-dependently, as shown by the test results obtained in the aboveTest Example 1.

Details of the experimental procedures in this Test Example 3 are asfollows.

In the same manner as in the above Test Example 1, the neovasculaturelesions comprising the experimental choroidal neovascular vessels (CNV)were produced in the ocular fundi of monkeys of the genus Macaca,according to the modified Ryan's method.

An aqueous solution of ICG dissolved in distilled water for theinjections at a concentration of 5.0 mg/ml of ICG was intravenouslyinjected at a dose of ICG of 0.5 mg/kg at a vein in the lower limbs ofthe monkeys having the neovasculature lesions in the retina of the eye.

Immediately after the time of the intravenous injection of ICG, theocular fundi of the monkeys were continuously observed under irradiationof the beam of laser light containing a light of 790 nm-wavelength bymeans of an infrared light camera apparatus (of a modified TCR 50-IAtype, manufactured by Topcon Co.) for infrared fluorescence fundus theangiography for diagnosing the ocular fundus.

At a time point of about 7 to 8 minutes after the time of theintravenous injection of ICG, the infrared fluorescence of ICG wasobserved at a prominent and appreciable intensity in the retinal normalvascular vessels, and at said time point, the ophthalmologist cancommence to observe that the infrared fluorescence of ICG was emittedalso in the choroidal neovascular vessels (CNV) at a slight butappreciable intensity. Subsequently, the intensity of the infraredfluorescence of ICG emitted in the CNV was increasing with the timelapse.

At the time point of 20 minutes after the time of the administration ofICG, the intensity of the infrared fluorescence of ICG in the retinalnormal vascular vessels had decreased, while the intensity of theinfrared fluorescence of ICG emitted in the CNV had increased to aprominent and appreciable intensity which was confirmable by theobserver.

TEST EXAMPLE 4

This Example illustrates an embodiment of the method of the secondaspect of this invention.

-   (a) The test monkeys having the experimental CNV in the subretinal    space of the ocular fundus, which were employed in the above Test    Example 3, were again used in this Test Example 4 as test animals.    To these test monkeys was intravenously injected ICG at a dose of 1    mg/kg in the same way as in Test Example 3. Immediately after the    time of the intravenous injection of ICG, the ocular fundi of the    monkeys were continuously observed by means of an infrared light    fundus camera apparatus (of modified TCR 50 type, manufactured by    Topcon Co., Ltd.) for the infrared fluorescence fundus angiography    for diagnosing the ocular fundus. The observation with the fundus    camera was made under irradiation of the ocular fundi with the laser    light containing a light of 790 nm-wavelength. There was recorded    the time point at which the infrared fluorescence of ICG was    eliminated entirely from the retinal normal vascular vessels. The    time-gap which was extended between the aforesaid time point as    recorded for the entire elimination of the infrared fluorescence of    IGC and the time of the intravenous injection of ICG was calculated    in a unit of “minutes” or “second”, whereby it was estimated that    said time-gap was 30 minutes.-   (b) Time was allowed to pass until the infrared fluorescence of ICG    was eliminated entirely from the neovasculature lesions in the    ocular fundi of the test monkeys. Thereafter, a volume of an aqueous    solution of mono-L-aspartyl chlorin e6 tetra-sodium salt (NPe6)    dissolved in physiological saline at a concentration of 5 mg/ml of    NPe6 was intravenously injected at a vein in the lower limbs of the    monkeys so that NPe6 was given at a dose of 1 mg/kg.-   (c) At a time point of 30 minutes after the time of the intravenous    injection of NPe6, it was started to irradiate a beam of laser light    of 664 nm-wavelength (as emitted from a semiconductor-type laser    generator, manufactured by Matsushita Industry System, Co. Ltd.,    Japan) in the form of a laser-irradiating spot of 1 mm diameter to 3    lesions of the neovasculature lesions under test. The 664 nm-laser    light as irradiated was irradiated at an irradiance of 0.57 W/cm₂    for 180 seconds so that the fluence was of 102.6 J/cm².

After the terminated irradiation of the 664 nm-laser light, the testmonkeys were fed under routine feeding conditions for one week.Thereafter, the effects of the PDT treatments were judged in the sameway as in the above Test Example 2, by the fluorescence fundusangiography with fluorescein and by the fluorescence fundus angiographywith ICG. It was found that the CNV present in the 3 neovasculaturelesions as treated with the laser irradiation were successfullyoccluded, while the retinal normal vascular vessels which were adjacentto the so treated neovasculature lesions, as well as the retinalparenchymal tissue and the choroidal normal vascular vessels present inthe surrounding regions in the retina could remain intact. Accordingly,it was found in this Example that the CNV present in the neovasculaturelesions could selectively be occluded, with success.

TEST EXAMPLE 5

This Example illustrates an embodiment of the method of the third aspectof this invention.

-   (a) Indocyanine green (ICG) was dissolved at a concentration of 10    mg/ml in a volume of physiological saline, and mono-L-aspartyl    chlorin e6 tetra-sodium salt (NPe6) was dissolved at a concentration    of 10 mg/ml in a further volume of physiological saline. The    resulting two solutions were mixed together in the equal    proportions, to prepare an aqueous solution usable for the    intravenous injection of ICG and NP6. Monkeys, which had the    experimental CNV in the subretinal space of the ocular fundus as    produced by the modified Ryan's method described in the above Test    Example 1, were used in this Test Example 5 as test animals.

The above-mentioned aqueous solution of ICG and NPe6 usable for theintravenous injection as prepared in the above was intravenouslyinjected to the test monkeys in the same way as in the above TestExample 3. It was prescribed that the dose of ICG was 0.5 mg/kg and thedose of NPe6 was 0.5 mg/kg upon the injection of them.

Immediately after the time of the intravenous injection of ICG and NPe6,the ocular fundi of the test monkeys were continuously photographed andobserved under irradiation of the laser light containing a light of 790nm-wavelength, by means of the infrared light fundus camera apparatus(of a modified TCR50-IA-type, manufactured by Topcon Co., Ltd.) for thefluorescence fundus angiography of diagnosing the ocular fundus, in thesame way as in Test Example 3.

At a time point of about 7 to 8 minutes after the time of theintravenous injection of ICG and NPe6, the infrared fluorescence of ICGwas observed at a prominent and appreciable intensity in the retinalnormal vascular vessels. At a time point of 20 minutes after the time ofthe intravenous injection of ICG and NPe6, it was observed that theinfrared fluorescence of ICG in the retinal normal vascular vessels haddecreased to a poor intensity, while the infrared fluorescence wasretained at a prominent and apreciable intensity in the vascular wallsof CNV present in the neovasculature lesions under observation.

-   (b) At said time point of 20 minutes after the time of the    intravenous injection of ICG and NPe6, the irradiation of the laser    light of 664 nm-wavelength was started in the form of    laser-irradiating spot of 1 mm diameter to 3 lesions of the    neovasculature lesions in the monkeys under test, in the same way as    in the above Test Example 4, (c). The 664 nm-laser light as    irradiated was irradiated at an irradiance of 0.68 W/cm² for 300    second so that the fluence was of 204 J/cm².

After the terminated irradiation of the 664-nm laser light, the testmonkeys were fed under routine feeding conditions for one week.Thereafter, the effects of the above PDT treatments were judged in thesame way as in the above Test Example 2, by the fluorescence fundusangiography with fluorescein and by the fluorescence fundus angiographywith indocyanine green (ICG). It was found that the CNV present in the 3neovasculature lesions comprising CNV as treated with the laserirradiation had been occluded successfully, while the retinal normalvascular vessels which were adjacent to the so treated neovasculaturelesions, as well as the retinal parenchymal tissue and the choroidalnormal vascular vessels present in the surrounding region in the retinacould remain intact. Accordingly, it was revealed also in this Examplethat the CNV present in the neovasculature lesions could selectively beoccluded with success.

Example 1 for Formulation

100 mg of mono-L-aspartyl chlorin e6 tetra-sodium salt was dissolved in4 ml of distilled water ready for injections. The resulting aqueoussolution was then adjusted to pH 7.4. After the aseptic filtrationthereof, the resulting sterilized solution was lyophilized in vials. Theresulting lyophilized preparation may be dissolved in an appropriatevolume of physiological saline, before its use.

Example 2 of Formulation

10 mg/ml of ICG was dissolved in a volume of sterile physiologicalsaline at a concentration of 10 mg/ml. Further, mono-L-aspartyl chlorine6 tetra-sodium salt was dissolved in a further volume of physiologicalsaline at a concentration of 10 mg/ml. The resultant two aqueoussolutions were mixed together in the equal proportions. The resultingsolution of ICG and NPe6 is suitable for the intravenous injection ofICG and NPe6 in order to carry out the method of the third aspect ofthis invention.

INDUSTRIAL APPLICABILITY

As described hereinbefore, in short, this invention provides a methodfor selectively occluding the neovascular vessels in the ocular fundus,which comprises intravenously administering mono-L-aspartyl chlorin e6or a salt thereof, particularly the tetra-sodium salt thereof at a doseof 0.5 to 10 mg/kg to a patient; subsequently estimating an appropriatetiming, that is, the time point when mono-L-aspartyl chlorin e6substance as administered has decreased in or has been eliminated fromthe retinal normal vascular vessels of the ocular fundus of the patientbut still has accumulated and is remaining at a significant andappreciable concentration in the vascular walls of the neovascularvessels in the ocular fundus; starting at an appropriate time point soestimated to irradiate a laser light of 664 nm-wavelength; andirradiating the 664 nm-laser light to the neovasculature lesionscomprising the neovascular vessels, at a certain controlled fluence.

By this invention, it is made feasible to occlude selectively suchneovascular vessels in the ocular fundus which can be formed in diseaseshaving the incidence of choroidal neovascular vessels, for example,age-related macular degeneration, and which can be formed in diseaseshaving the incidence of retinal neovascular vessels, for example,proliferative diabetic retinitis. Thus, this invention is useful fortherapeutic treatment of said ophthalmological diseases.

1. A photodynamic therapy method for selectively occluding vesselscomprising choroidal neovascular vessels and retinal neovascular vesselsformed in a mammal having an ocular fundus tissue comprising retinalnormal parenchymal tissue, retinal normal vascular vessels, choroidalnormal vascular vessels lying under the retina, choroidal neovascularvessels and retinal neovascular vessels, said method comprising: (a)administering intravenously a photosensitizer comprising a chlorin e6compound selected from the group consisting of a mono-L-aspartyl chlorine6 and a pharmaceutically acceptable salt thereof, to the mammal at adose of 0.5 mg/kg to 10 mg/kg calculated on the weight basis of thephotosensitizer; (b) allowing the photosensitizer to be carried along bythe blood stream circulating in the ocular retinal central artery andciliary artery, to be taken up by the endothelial cell layers of theretinal normal vascular vessels, the endothelial cell layers of thechoroidal normal vascular vessels, and the endothelial cell layers ofthe retinal neovascular vessels and/or the endothelial cell layers ofthe choroidal neovascular vessels, and allowing the photosensitizer tobe distributed and accumulated in and being eliminated or cleared fromthe vascular walls of the normal vascular vessels and the neovascularvessels; (c) subsequently irradiating and scanning, using a laser lightat a 488 nm wavelength at a sufficient power, intermittently orcontinuously, through the surface of ocular cornea, pupil, lens andvitreous fluid of the eye of the mammal, to and on the ocular fundustissue of the mammal, so as to photo-excite the photosensitizer, therebyinducing emission of a red fluorescence from the now photo-excitedphotosensitizer having been distributed in the vascular walls of thenormal vascular vessels and in the vascular walls of the neovascularvessels in the ocular fundus tissue; and further observingintermittently or continuously the intensity of the red fluorescencewhich is emitted from the photo-excited photosensitizer by using afluorescence ocular fundus angiography apparatus or a fluorescencemicroscope for funduscopy; (d) using the fluorescence ocular fundusangiography apparatus or fluorescence microscope for funduscopy duringthe irradiation of the 488 nm-wavelength laser light, to estimate a timepoint at which, in the course of the elimination or clearance of thephotosensitizer out of the vascular walls of the retinal normal vascularvessels and the choroidal normal vascular vessels, the intensity of thered fluorescence emitted from the photosensitizer present in thevascular walls of the retinal normal vascular vessels under observationcan be observed to have decreased to a lowered value of aboutone-half-fold or less of the intensity of the red fluorescence emittedfrom the photosensitizer remaining in the vascular walls of theneovascular vessels under observation; and thus establishing anestimated time point when the concentration of the photosensitizerpresent in the vascular walls of the retinal normal vascular vesselsunder observation has decreased to a lowered value of aboutone-half-fold or less; (e) at the estimated time point provided in saidstep (d) or within 1 to 10 minutes from the estimated time point;starting to irradiate a laser light of 664 nm-wavelength in the form ofa thin laser beam, via the cornea, pupil and vitreous fluid of the eye,exclusively to targeted lesions comprising the neovasculature formed ofthe neovascular vessels in the ocular fundus, in such a way that thelaser light of 664 nm-wavelength is irradiated at a power necessary toexcite the photosensitizer remaining in the vascular walls of theneovascular vessels; and (f) subsequently permitting the lumens of theneovascular vessels contained in the lesions as irradiated with thelaser light of 664 nm-wavelength to be occluded by the developed actionsof the photochemical reaction of the laser-excited photosensitizerremaining in the vascular walls of the neovascular vessels, whereby aselective occlusion of the choroidal neovascular vessels and/or theretinal neovascular vessels is achieved.
 2. The method according toclaim 1, wherein, in said step (e) of the method, the irradiation usingthe laser light of 664 nm-wavelength is started at a time point when atime period of 10 to 70 minutes has lapsed after the time of theintravenous injection of the photosensitizer; and said time point isalso at least the estimated time point determined in step (d).
 3. Themethod according to claim 1, wherein, the estimated time pointdetermined in said step (d) is 20 to 70 minutes from the time of theintravenous injection of the photosensitizer; and that said estimatedtime point is also such time point when it can be determined from theobservation with the fluorescence ocular fundus angiography apparatus orfluorescence microscope for funduscopy during the irradiation of the 488nm-wavelength laser light, that the intensity of the red fluorescenceemitted from the photosensitizer remaining in the vascular walls of thechoroidal neovascular vessels and/or the retinal neovascular vessels hasreached its peak value under the irradiation of the laser light of 488nm-wavelength; and that said estimated time point is further such timepoint when it is additionally observed by said fluorescence ocularfundus angiography apparatus or fluorescence microscope, that the redfluorescence as emitted from the photosensitizer still remaining in thevascular walls of the retinal normal vascular vessels has disappearedcompletely during the irradiation of the laser light of 488nm-wavelength, and wherein, at the estimated time point, the laser lightof 664 nm-wavelength is then started in said step (e) exclusivelytargeting lesions comprising the neovasculature formed of theneovascular vessels in the ocular fundus.
 4. A method according to claim1, wherein the laser light of 664 nm-wavelength, which is irradiated tothe targeted lesions comprising the neovascular vessels in the ocularfundus, has an irradiance of 10 to 1500 mW/cm², on the top retina face,as measured at the corneal surface by means of an optical power meter,and said laser light of 664 nm-wavelength is irradiated for a durationof 10 to 300 seconds and at a fluence of the laser light in a range of7.0 J/cm² to 250 J/cm², provided that said fluence of the laser light isevaluated by multiplying the laser irradiance (in W/cm²) by the durationof the laser irradiation (in seconds), and wherein the lower is the doseof mono-L-aspartyl chlorin e6, the fluence of the laser light to beirradiated is controlled to be the higher, so as to achieve theselective occlusion of the neovascular vessels present in the targetedlesions.
 5. A method according to claim 1, wherein the laser light of664 nm-wavelength is started in the step (e) of the method to beirradiated at such a time point when a time period of 20 to 70 minuteshas lapsed after the time of the intravenous administration ofmono-L-aspartyl chlorin e6 or a salt thereof, particularlymono-L-aspartyl chlorin e6 tetra-sodium salt, as long as the chlorin e6compound is given at a dose of 0.5 mg/kg to 10.0 mg/kg (as calculated onthe weight basis of mono-L-aspartyl chlorin e6 tetra-sodium salt, namelyNPe6; and the same way of this calculation is applied hereinafter), andwherein the fluence of the laser light of 664 nm-wavelength to beirradiated is controlled within a range of 7 J/cm² to 205 J/cm².
 6. Amethod according to claim 1, wherein the laser light of 664nm-wavelength is started in the step (e) of the method to be irradiatedat such a time point when a time period of 20 to 30 minutes has lapsedafter the time of the intravenous administration of mono-L-aspartylchlorin e6 or a salt thereof, particularly mono-L-aspartyl chlorin e6tetra-sodium salt, as long as the chlorin e6 compound is given at a doseof 0.5 mg/kg to 0.9 mg/kg (as calculated on the weight basis of NPe6),and wherein the fluence of the laser light of 664 nm-wavelength to beirradiated is controlled within a range of 175 J/cm² to 205 J/cm².
 7. Amethod according to claim 1, wherein the laser light of 664nm-wavelength is started in the step (e) of the method to be irradiatedat such a time point when a time period of 30 to 60 minutes has lapsedafter the time of the intravenous administration of mono-L-aspartylchlorin e6 or a salt thereof, as long as the chlorin e6 compound isgiven at a dose of 1 mg/kg to 1.9 mg/kg (as calculated on the weightbasis of NPe6), and wherein the fluence of the laser light of 664nm-wavelength to be irradiated is controlled within a range of 30 J/cm²to 175 J/cm².
 8. A method according to claim 1, wherein the laser lightof 664 nm-wavelength is started in the step (e) of the method to beirradiated at such a time point when a time period of 60 minutes haslapsed after the time of the intravenous administration ofmono-L-aspartyl chlorin e6 or a salt thereof, particularlymono-L-aspartyl chlorin e6 tetra-sodium salt, as long as the chlorin e6compound is given at a dose of 2 mg/kg to 9.5 mg/kg (as calculated onthe weight basis of NPe6), and wherein the fluence of the laser light of664 nm-wavelength to be irradiated is controlled within a range of 30J/cm² to 45 J/cm².
 9. A method according to claim 1, wherein the laserlight of 664 nm-wavelength is started in the step (e) of the method tobe irradiated at such a time point when a time period of 60 to 70minutes has lapsed after the time of the intravenous administration ofmono-L-aspartyl chlorin e6 or a salt thereof, as long as the chlorin e6compound is given at a dose of 9.5 mg/kg to 10 mg/kg (as calculated onthe weight basis of NPe6), and wherein the fluence of the laser light of664 nm-wavelength to be irradiated is controlled within a range of 7J/cm² to 9 J/cm².
 10. A method according to claim 1, wherein themammalian animal to be treated is a human having suffered fromage-related macular degeneration with the choroidal neovascular vessels.11. A method according to claim 1, wherein the mammalian animal to betreated is a human having suffered from proliferative diabetic retinitiswith proliferative neovascular vessels in the retina.
 12. A photodynamictherapy method for selectively occluding various ocular vesselscomprising choroidal neovascular vessels, and/or retinal neovascularvessels formed in a mammal having an ocular fundus tissue comprisingretinal normal parenchymal tissue, retinal normal vascular vessels,choroidal normal vascular vessels lying under the retina, and choroidalneovascular vessels and retinal neovascular vessels, said methodcomprising: (a) administering initially indocyanine green intravenouslyto the mammal at a dose of 0.5 mg/kg to 1 mg/kg; (b) allowing theindocyanine green to be carried along by the blood stream circulating inthe ocular retinal central artery and ciliary artery, to be uptaken inthe endothelial cell layers of the retinal normal vascular vessels, inthe endothelial cell layers of the choroidal normal vascular vessels, inthe endothelial cell layers of the choroidal neovascular vessels and/orthe endothelial cell layers of the retinal neovascular vessels, andallowing the indocyanine green to be distributed and accumulated in andbeing eliminated or cleared from the vascular walls of the normalvascular vessels and the neovascular vessels; (c) subsequentlyirradiating and scanning using a laser light having a 790 nm-wavelengthat a sufficient power, intermittently or continuously, through thesurface of ocular cornea, pupil, lens and vitreous fluid of the eye, toand on the ocular fundus tissue of the mammal, so as to photo-excite theindocyanine green inducing emission of an infrared fluorescence from thenow photo-excited indocyanine green having been distributed in thevascular walls of the normal vascular vessels and in the vascular wallsof the neovascular vessels in the ocular fundus tissue; and furtherobserving intermittently or continuously the intensity of the infraredfluorescence emitted from the now photo-excited indocyanine green usinga fluorescence ocular fundus angiography apparatus or a fluorescencemicroscope for funduscopy; (d) using the fluorescence ocular fundusangiography apparatus or fluorescence microscope during the irradiationof the 790 nm-laser light of said step (d), to estimate a time point atwhich, in the course of the elimination or clearance of indocyaninegreen out of the vascular walls of the retinal normal vascular vesselsand the choroidal normal vascular vessels, the intensity of the infraredfluorescence emitted from the indocyanine green present in the vascularwalls of the retinal normal vascular vessels under observation can beobserved to have decreased to a lowered value of about one-half-fold orless of the intensity of the infrared fluorescence emitted from theindocyanine green remaining in the vascular walls of the neovascularvessels under observation; and thus establishing an estimated time pointat which the concentration of the indocyanine green present in thevascular walls of the retinal normal vascular vessels under observationhas decreased to a lowered value of about one-half-fold or less; (e)calculating a time-gap extending between the time of the firstintravenous injection of indocyanine green and the estimated time pointdetermined in said step (d); (f) allowing time to pass until it can beobserved that the infrared fluorescence of the indocyanine greenremaining in the vascular walls of the choroidal neovascular vesselsand/or retinal neovascular vessels has disappeared completely; (g)administering a photosensitizer comprising a chlorin e6 compoundselected from the group consisting of mono-L-aspartyl chlorin e6 and apharmaceutically acceptable salt thereof to the mammal intravenously ata dose in a range of 0.5 mg/kg to 10 mg/kg as calculated on the weightbasis of NPe6, after said step (f); (h) allowing the chlorin e6 compoundto be carried along by the blood stream circulating in the ocularretinal central artery and ciliary artery, to be uptaken in theendothelial cell layers of the retinal normal vascular vessels, theendothelial cell layers of the choroidal normal vascular vessels, theendothelial cell layers of the choroidal neovascular vessels and/or theendothelial cell layers of the retinal neovascular vessels, and allowingthe administered chlorin e6 compound to be distributed and accumulatedin and being eliminated or cleared from the vascular walls of the normalvascular vessels and of the neovascular vessels; (i) permitting theadministered chlorin e6 compound to be accumulated and remain in thevascular walls of the choroidal neovascular vessels and/or the retinalneovascular vessels, while the chlorin e6 photosensitizer compound isconcurrently eliminated or cleared out of the vascular walls of thenormal vascular vessels of the retina and choroid; (j) at a time pointwhen a time duration, which has a time length equal to that of thetime-gap calculated in said step (e), has just passed after the time ofthe intravenous injection of the chlorin e6 compound; starting toirradiate using a laser light of 664 nm-wavelength in the form of a thinlaser beam, via the surface of cornea, pupil and vitreous fluid of theeye, exclusively to targeted lesions comprising the neovasculatureformed of the neovascular vessels in the ocular fundus, in such a waythat the laser light of 664 nm-wavelength is irradiated at a powernecessary to excite the photosensitizer remaining in the vascular wallsof the neovascular vessels; and (k) subsequently permitting the lumensof the neovascular vessels contained in the lesions irradiated with thelaser light of 664 nm-wavelength to be occluded by the developed actionsof the photochemical reaction of the laser-excited photosensitizerremaining in the vascular walls of the neovascular vessels, whereby aselective occlusion of the choroidal neovascular vessels and/or theretinal neovascular vessels is achieved.
 13. A method according to claim12, wherein, in the step (j) of the method, the irradiation of the laserlight of 664 nm-wavelength via the surface of cornea, pupil and vitreousfluid of the eye exclusively to the targeted lesions comprising theneovasculatures formed of the neovascular vessels in the ocular fundusis started at such a time point when a time period of 10 to 70 minutes,has lapsed after the time of the intravenous administration ofmono-L-aspartyl chlorin e6 or a salt thereof, provided that said timepoint is the such time point when there has just passed a time durationwhich has a time length equal to that of the said time-gap as calculatedin the step (e) of the method according to claim
 12. 14. A methodaccording to claim 12, wherein the laser light of 664 nm-wavelength,which is irradiated to the targeted lesions comprising the neovascularvessels in the ocular fundus, has an irradiance of 10 to 1500 mW/cm², onthe top retina face, as measured at the corneal surface by means of anoptical power meter, and the laser light of 664 nm-wavelength isirradiated for a duration of 10 to 300 second, and at a fluence of thelaser light in a range of 7.0 J/cm² to 250 J/cm², provided that saidfluence of the laser light is evaluated by multiplying the laserirradiance (in W/cm²) by the duration of the laser irradiation (inseconds), and wherein the lower is the dose of mono-L-aspartyl chlorine6, the fluence of the laser light of 664 nm-wavelength to be irradiatedis controlled to be the higher, so as to achieve the selective occlusionof the neovascular vessels present in the target lesions.
 15. A methodaccording to claim 12, wherein the irradiation of the laser light of 664nm-wavelength is started in the step (j) of the method at such a timepoint when a time period of 20 to 70 minutes has lapsed after the timeof the intravenous administration of mono-L-aspartyl chlorin e6 or asalt thereof, particularly mono-L-aspartyl chlorin e6 tetra-sodium salt,as long as the chlorin e6 compound is given at a dose of 0.5 mg/kg to10.0 mg/kg (as calculated on the weight basis of NPe6), and wherein thefluence of the laser light of 664 nm-wavelength to be irradiated iscontrolled within a range of 7 J/cm² to 205 J/cm².
 16. A methodaccording to claim 12, wherein the irradiation of the laser light of 664nm-wavelength is started in the step (j) of the method at such a timepoint when a time period of 20 to 30 minutes has lapsed after the timeof the intravenous administration of mono-L-aspartyl chlorin e6 or asalt thereof, particularly mono-L-aspartyl chlorin e6 tetra-sodium salt,as long as the chlorin e6 compound is given at a dose of 0.5 mg/kg to0.9 mg/kg (as calculated on the weight basis of NPe6), and wherein thefluence of the laser light of 664 nm-wavelength to be irradiated iscontrolled within a range of 175 J/cm² to 205 J/cm².
 17. A methodaccording to claim 12, wherein the irradiation of the laser light of 664nm-wavelength is started in the step (j) of the method at such a timepoint when a time period of 30 to 60 minutes has lapsed after theintravenous administration of mono-L-aspartyl chlorin e6 or a saltthereof, as long as the chlorin e6 compound is given at a dose of 1mg/kg to 1.9 mg/kg (as calculated on the weight basis of NPe6), andwherein the fluence of the laser light of 664 nm-wavelength to beirradiated is controlled within a range of 30 J/cm² to 175 J/cm².
 18. Amethod according to claim 12, wherein the irradiation of the laser lightof 664 nm-wavelength is started in the step (j) of the method at such atime point when a time period of 60 minutes has lapsed after the time ofthe intravenous administration of mono-L-aspartyl chlorin e6 or a saltthereof, particularly mono-L-aspartyl chlorin e6 tetra-sodium salt, aslong as the chlorin e6 compound is given at a dose of 2 mg/kg to 9.5mg/kg (as calculated on the weight basis of NPe6), and wherein thefluence of the laser light of 664 nm-wavelength to be irradiated iscontrolled within a range of 30 J/cm² to 45 J/cm².
 19. A methodaccording to claim 12, wherein the irradiation of the laser light of 664nm-wavelength is started in the step (j) of the method at such a timepoint when a time period of 60 to 70 minutes has lapsed after the timeof the intravenous administration of mono-L-aspartyl chlorin e6 or asalt thereof, particularly mono-L-aspartyl chlorin e6 tetra-sodium salt,as long as the chlorin e6 compound is given at a dose of 9.5 mg/kg to 10mg/kg (as calculated on the weight basis of NPe6), and wherein thefluence of the laser light of 664 nm-wavelength to be irradiated iscontrolled within a range of 7 J/cm² to 9 J/cm².
 20. A method accordingto claim 12, wherein the mammalian animal to be treated is a humanhaving suffered from age-related macular degeneration with the choroidalneovascular vessels.
 21. A method according to claim 12, wherein themammalian animal to be treated is a human having suffered fromproliferative diabetic retinitis with proliferative neovascular vessels.22. A photodynamic therapy method for selectively occluding ocularvessels formed in a mammal having ocular fundus tissue and comprisingretinal normal parenchymal tissue, retinal normal vascular vessels andchoroidal normal vascular vessels lying under the retina, choroidalneovascular vessels and retinal neovascular vessels, said methodcomprising: (a) administering intravenously a photosensitizer comprisinga chlorin e6 compound selected from the group consisting ofmono-L-aspartyl chlorin e6 and a pharmaceutically acceptable saltthereof, to the mammal at a dose of 0.5 mg/kg to 10 mg/kg calculated onthe weight basis of the chlorin e6 compound; and injecting intravenouslyindocyanine green to the mammal at a dose in a range of 0.5 mg/kg to 1mg/kg, at the same time as or immediately before or after saidintravenous administration of the chlorin e6 compound; (b) allowing thechlorin e6 compound and the indocyanine green to be carried along by theblood stream circulating in the ocular retinal central artery andciliary artery of the mammal, to be uptaken in the endothelial celllayers of the retinal normal vascular vessels, the endothelial celllayers of the choroidal normal vascular vessels, in the endothelial celllayers of the retinal neovascular vessels, and/or the endothelial celllayers of the choroidal neovascular vessels, and allowing the chlorin e6compound and indocyanine green to be distributed and accumulated in andbeing eliminated or cleared from the vascular walls of the normalvascular vessels and of the said neovascular vessels; (c) subsequentlyirradiating and scanning using a laser light at a 790 nm wavelength,intermittently or continuously, through the surface of ocular cornea,pupil, lens and vitreous fluid of the eye, to and on the ocular fundustissue of the animal, so as to induce emission of an infraredfluorescence from the indocyanine green selectively, in the co-presenceof the chlorin e6 compound and indocyanine green having been distributedin the vascular walls of the vascular vessels of the ocular fundustissue; and observing intermittently or continuously, the intensity ofinfrared fluorescence emitted from the indocyanine green distributed inthe vascular walls of the retinal normal vascular vessels, the vascularwalls of the choroidal neovascular vessels and/or the vascular walls ofthe retinal neovascular vessels, using a fluorescence ocular fundusangiography apparatus or a fluorescence microscope for funduscopy; (d)using the fluorescence ocular fundus angiography apparatus orfluorescence microscope during the irradiation at 790 nm-wavelength, toestimate a time point at which, in the course of the eliminations orclearances of the chlorin e6 compound and indocyanine green from thevascular walls of the retinal normal vascular vessels and the choroidalnormal vascular vessels, the intensity of the infrared fluorescence asemitted from the indocyanine green present in the vascular walls of theretinal normal vascular vessels under observation can be observed tohave decreased to a lowered value of about one-half-fold or less of theintensity of the infrared fluorescence as emitted from the indocyaninegreen remaining in the vascular walls of the neovascular vessels underobservation; and thus establishing an estimated time point at which theconcentration of the chlorin e6 compound present in the vascular wallsof the retinal normal vascular vessels under observation has decreasedto a lowered value of about one-half-fold or less; (e) at the estimatedtime point determined in said step (d) or within 1–10 minutes from theestimated time point; starting to irradiate using a laser light at a 664nm wavelength in the form of a thin laser beam, via the surface ofcornea, pupil and vitreous fluid of the eye, exclusively to targetedlesions comprising neovasculature formed of the neovascular vessels inthe ocular fundus, in such a way that the laser light at 664 nmwavelength is irradiated at a power necessary to excite the chlorin e6compound remaining in the vascular walls of the neovascular vessels; and(f) subsequently permitting that the lumens of the neovascular vesselscontained in the lesions irradiated with the laser light at 664 nmwavelength to be occluded by developed actions of a photochemicalreaction of the laser-excited chlorin e6 compound remaining in thevascular walls of the neovascular vessels, whereby a selective occlusionof the choroidal neovascular vessels and/or the retinal neovascularvessels is achieved.
 23. The method according to claim 22, wherein, saidstep (e) is started at a time point when a time period of 10 to 70minutes has lapsed after the time of the intravenous administration ofthe chlorin e6 compound where said time point is also the estimated timepoint determined in said step (d).
 24. The method according to claim 22,wherein, in said step (d), the estimated time point is 20 to 70 minutesfrom the time of the intravenous injection of the chlorin e6 compoundand the estimated time point is a time point when it can be revealedfrom the observation with the fluorescence ocular fundus angiographyapparatus or fluorescence microscope for funduscopy that the intensityof the infrared fluorescence emitted from the indocyanine greenremaining in the vascular walls of the choroidal neovascular vesselsand/or the retinal neovascular vessels has reached its peak value underthe irradiation of the laser light at 790 nm-wavelength; and theestimated time point is a time point when it is also observed by thefluorescence ocular fundus angiography apparatus or fluorescencemicroscope for funduscopy that the infrared fluorescence as emitted fromthe indocyanine green still remaining in the vascular walls of theretinal normal vascular vessels has disappeared completely even underthe irradiation of the laser light at 790 nm-wavelength, and wherein atthe estimated time point, said step (e) is initiated.
 25. A methodaccording to claim 22, wherein the laser light of 664 nm-wavelength,which is irradiated to the targeted lesions comprising the neovascularvessels in the ocular fundus, has an irradiance of 10 to 1500 mW/cm², onthe top retina face, as measured at the corneal surface by means of anoptical power meter, and said laser light of 664 nm-wavelength isirradiated for a duration of 10 to 300 seconds and at a fluence of thelaser light in a range of 7.0 J/cm² to 250 J/cm², provided that saidfluence of the laser light is evaluated by multiplying the laserirradiance (in W/cm²) by the duration of the laser irradiation (inseconds), and wherein the lower is the dose of mono-L-aspartyl chlorine6, the fluence of the laser light of 664 nm to be irradiated iscontrolled to be the higher, so as to achieve the selective occlusion ofthe neovascular vessels present in the targeted lesions.
 26. A methodaccording to claim 22, wherein the laser light of 664 nm-wavelength isstarted in the step (e) of the method to be irradiated at such a timepoint when a time period of 20 to 70 minutes has lapsed after the timeof the intravenous administration of mono-L-aspartyl chlorin e6 or asalt thereof, particularly mono-L-aspartyl chlorin e6 tetra-sodium salt,as long as the chlorin e6 compound is given at a dose of 0.5 mg/kg to10.0 mg/kg (as calculated on the weight basis of NPe6), and wherein thefluence of the laser light of 664 nm-wavelength to be irradiated iscontrolled within a range of 7 J/cm² to 205 J/cm².
 27. A methodaccording to claim 22, wherein the laser light of 664 nm-wavelength isstarted in the step (e) of the method to be irradiated at such a timepoint when a time period of 20 to 30 minutes has lapsed after the timeof the intravenous administration of mono-L-aspartyl chlorin e6 or asalt thereof, particularly mono-L-aspartyl chlorin e6 tetra-sodium salt,as long as the chlorin e6 compound is given at a dose of 0.5 mg/kg to0.9 mg/kg (as calculated on the weight basis of NPe6), and wherein thefluence of the laser light of 664 nm-wavelength to be irradiated iscontrolled within a range of 175 J/cm² to 205 J/cm².
 28. A methodaccording to claim 22, wherein the laser light of 664 nm-wavelength isstarted in the step (e) of the method to be irradiated at such a timepoint when a time period of 30 to 60 minutes has lapsed after the timeof the intravenous administration of mono-L-aspartyl chlorin e6 or asalt thereof, as long as the chlorin e6 compound is given at a dose of 1mg/kg to 1.9 mg/kg (as calculated on the weight basis of NPe6), andwherein the fluence of the laser light of 664 nm-wavelength to beirradiated is controlled within a range of 30 J/cm² to 175 J/cm².
 29. Amethod according to claim 22, wherein the laser light of 664nm-wavelength is started in the step (e) of the method to be irradiatedat such a time point when a time period of 60 minutes has lapsed afterthe time of the intravenous administration of mono-L-aspartyl chlorin e6or a salt thereof, as long as the chlorin e6 compound is given at a doseof 2 mg/kg to 9.5 mg/kg (as calculated on the weight basis of NPe6), andwherein the fluence of the laser light of 664 nm-wavelength to beirradiated is controlled within a range of 30 J/cm² to 45 J/cm².
 30. Amethod according to claim 22, wherein the laser light of 664nm-wavelength is started in the step (e) of the method to be irradiatedat such a time point when a time period of 60 to 70 minutes has lapsedafter the time of the intravenous administration of mono-L-aspartylchlorin e6 or a salt thereof, as long as the chlorin e6 compound isgiven at a dose of 9.5 mg/kg to 10 mg/kg (as calculated on the weightbasis of NPe6), and wherein the fluence of the laser light of 664nm-wavelength to be irradiated is controlled within a range of 7 J/cm²to 9 J/cm².
 31. A method according to claim 22, wherein the mammaliananimal to be treated is a human having suffered from age-related maculardegeneration with the choroidal neovascular vessels.
 32. A methodaccording to claim 22, wherein the mammalian animal to be treated is ahuman having suffered from proliferative diabetic retinitis withproliferative neovascular vessels in the retina.
 33. The photodynamictherapy method of claim 1, wherein the chlorin e6 compound comprisesmono-L-aspartyl chlorin e6 tetra-sodium salt.
 34. The method of claim 12wherein the chlorin e6 compound comprises mono-L-aspartyl chlorin e6tetra-sodium salt.
 35. The method of claim 22 wherein the chlorin e6compound comprises mono-L-aspartyl chlorin e6 tetra-sodium salt.
 36. Themethod of claim 1 wherein in said step (d) the estimated time point isdetermined as the time when it is observed that the intensity of the redfluorescence emitted from the photosensitizer has decreased to ⅓-fold orless.
 37. The method of claim 12 wherein in said step (d), the estimatedtime is determined as the time point when it is observed that theintensity of the infrared fluorescence emitted from the indocyaninegreen has decreased to ⅓ fold or less in intensity.
 38. The method ofclaim 22 wherein in said step (d), the estimated time period is a timepoint when it is observed that the intensity of infrared fluorescenceemitted from the indocyanine green has decreased to ⅓-fold or less inintensity.